Respiratory syncytial virus (rsv) vaccine

ABSTRACT

The present invention relates to an mRNA sequence, comprising a coding region, encoding at least one antigenic peptide or protein of RSV infections Respiratory syncytial virus (RSV) or a fragment, variant or derivative thereof. Additionally the present invention relates to a composition comprising a plurality of mRNA sequences comprising a coding region, encoding at least one antigenic peptide or protein of RSV infections Respiratory syncytial virus (RSV) or a fragment, variant or derivative thereof. Furthermore it also discloses the use of the mRNA sequence or the composition comprising a plurality of mRNA sequences for the preparation of a pharmaceutical composition, especially a vaccine, e.g. for use in the prophylaxis or treatment of RSV infections Respiratory syncytial virus (RSV) infections. The present invention further describes a method of treatment or prophylaxis of RSV infections using the mRNA sequence.

The present application is a divisional of U.S. application Ser. No.17/316,834, filed May 11, 2021, which is a continuation of U.S.application Ser. No. 16/168,747, filed Oct. 23, 2018, now U.S. Pat. No.11,034,729, which is a continuation of U.S. application Ser. No.15/488,815, filed Apr. 17, 2017, now U.S. Pat. No. 10,150,797, which isa continuation of U.S. application Ser. No. 15/048,439, filed Feb. 19,2016, now U.S. Pat. No. 9,688,729, which is a continuation ofInternational Application No. PCT/EP2014/002301, filed Aug. 21, 2014,the entire text of each of the above referenced disclosures beingspecifically incorporated herein by reference. International ApplicationNo. PCT/EP2014/002301 claims priority benefit of European ApplicationNo. PCT/EP2013/002518, filed Aug. 21, 2013.

BACKGROUND OF THE INVENTION

This application contains a Sequence Listing XML, which has beensubmitted electronically and is hereby incorporated by reference in itsentirety. Said Sequence Listing XML, created on Jul. 3, 2023, is namedCRVCP0151USD1.xml and is 63,816 bytes in size.

The present invention relates to an mRNA sequence, comprising a codingregion, encoding at least one antigenic peptide or protein ofRespiratory syncytial virus (RSV) or a fragment, variant or derivativethereof. Additionally the present invention relates to a compositioncomprising a plurality of mRNA sequences comprising a coding region,encoding at least one antigenic peptide or protein of Respiratorysyncytial virus (RSV) or a fragment, variant or derivative thereof.

Furthermore it also discloses the use of the mRNA sequence or thecomposition comprising a plurality of mRNA sequences for the preparationof a pharmaceutical composition, especially a vaccine, e.g. for use inthe prophylaxis or treatment of RSV infections. The present inventionfurther describes a method of treatment or prophylaxis of RSV infectionsusing the mRNA sequence.

The global medical need and economic impact of RSV is very high. It isthe most important cause of acute lower respiratory tract infections(ALRIs) that result in hospital visits during infancy and earlychildhood. For example, in the United States, more than 60% of infantsare infected by RSV during their first RSV season, and nearly all havebeen infected by 2-3 years of age. Approximately 2.1 million US childrenless than 5 years of age are treated for RSV disease each year: 3% asinpatients, 25% in emergency departments, and 73% in pediatricpractices. Globally, among children less than five years of age, RSVcauses an estimated 33.8 million ALRIs each year (more than 22% of allALRIs), resulting in 66 000-199 000 deaths, 99% of which occur indeveloping countries. RSV is also a common cause of respiratory diseaseamong the elderly, resulting in as many hospitalizations as influenza ina heavily influenza-immunized population. RSV spreads by respiratorydroplets and close contact with infected persons or contaminatedobjects. In temperate climates, there is an annual winter epidemic.Infants are at highest risk for severe RSV disease in their first 6months, and hospitalization peaks at 2-3 months of age. Preterm birthand cardiopulmonary disease are risk factors for severe RSV disease. RSVinfection of infants elicits partially protective immunity, whichappears to wane more rapidly than immunity against most otherrespiratory viruses. Most children infected with RSV during their firstyear are re-infected the next year, generally with less severe disease.Re-infections continue throughout life, often with upper respiratorytract symptoms, and sometimes with lower respiratory tract or sinusinvolvement. Recommended treatment of RSV bronchiolitis consistsprimarily of respiratory support and hydration. No specific anti-viraltherapy is recommended. The neutralizing monoclonal antibody Palivizumabis used for prophylaxis of infants at highest risk for severe infectionbut is too expensive and impractical for universal use. Currently, thereis no licensed RSV vaccine, and developing a safe and effective RSVvaccine is a global public health priority.

In a vaccine trial in the 1960s, infants and young children wereimmunized with a formalin-inactivated whole virion RSV preparation(FIRSV) or an equivalent paramyxovirus preparation (FIPIV). Five percentof the subjects who were immunized with FI-PIV and then naturallyinfected by RSV during the next RSV season were hospitalized; 80% ofthose who were immunized with FI-RSV and then infected by RSV werehospitalized, and two children died. This enhancement of an RSVinfection due to vaccination is a specific problem for the developmentof vaccines against RSV infections (reviewed in Shaw et al. Curr OpinVirol. 2013 June; 3(3):332-42. doi: 10.1016/j.coviro.2013.05.003. Epub2013 May 30.).

Therefore, Respiratory syncytial virus (RSV) infections are the greatestremaining unmet infant vaccine need in developed countries and animportant unmet infant vaccine need worldwide. More than 40 years ofeffort have not yet resulted in a licensed RSV vaccine for humans.

In summary, RSV which belongs to the virus family of Paramyxoviridae, isone of the most contagious pathogens and makes a substantialcontribution to severe respiratory tract infections in infants, theelderly and immunocompromised patients.

As mentioned above, currently a humanised monoclonal antibody againstthe viral surface F protein is the only prophylactic product on themarket which is recommended for infants considered at high riskincluding pre-term infants and infants with chronic lung disease (TheIMpact-RSV Study Group. 1998. Palivizumab, a Humanized RespiratorySyncytial Virus Monoclonal Antibody, Reduces Hospitalization FromRespiratory Syncytial Virus Infection in High-risk Infants. Pediatrics,102(3), S.531-537, Tablan et al. 2003. Guidelines for preventinghealth-care-associated pneumonia, 2003: recommendations of CDC and theHealthcare Infection Control Practices Advisory Committee. MMWR.Recommendations and Reports: Morbidity and Mortality Weekly Report.Recommendations and Reports/Centers for Disease Control, 53(RR-3),S.1-36.).

Recent studies with animal models demonstrated that sufficient amountsof neutralising antibodies targeting RSV F protein limit viralreplication leading to a less severe course of disease (Singh, S. R. etal., 2007. Immunogenicity and efficacy of recombinant RSV-F vaccine in amouse model. Vaccine, 25(33), S.6211-6223, Zhan, X. et al., 2007.Respiratory syncytial virus (RSV) F protein expressed by recombinantSendai virus elicits B-cell and T-cell responses in cotton rats andconfers protection against RSV subtypes A and B. Vaccine, 25(52),S.8782-8793, Vaughan, K., et al., 2005. DNA immunization againstrespiratory syncytial virus (RSV) in infant rhesus monkeys. Vaccine,23(22), S.2928-2942).

Moreover, it could be shown that a balanced regulatory and effector Tcell function is required for viral clearance and reduction of severityof illness (Liu, J. et al., 2010. Epitope-specific regulatory CD4 Tcells reduce virus-induced illness while preserving CD8 T-cell effectorfunction at the site of infection. Journal of Virology, 84(20),S.10501-10509).

Despite the above mentioned humanised monoclonal antibody,live-attenuated vaccine viruses were developed which elicit a strongimmune response, but which are not recommended for use in the specifictarget groups (infants, children, the elderly and immunocompromisedpatients). Also, DNA vectors expressing RSV F protein which bears B-cellepitopes were used to induce the production of neutralizing antibodies.In this context, WO 2008/077527 and WO 96/040945 disclose vectorscomprising DNA sequences encoding RSV F protein for the use as vaccines.However, the use of DNA as a vaccine may be dangerous due to unwantedinsertion into the genome, possibly leading to interruption offunctional genes and cancer or the formation of anti-DNA antibodies.

Therefore it is the object of the underlying invention to provide anmRNA sequence coding for antigenic peptides or proteins of Respiratorysyncytial virus (RSV) for the use as vaccine for prophylaxis ortreatment of RSV infections, particularly in infants, the elderly andimmunocompromised patients.

These objects are solved by the subject matter of the attached claims.Particularly, the objects underlying the present invention are solvedaccording to a first aspect by an inventive mRNA sequence comprising acoding region, encoding at least one antigenic peptide or protein ofRespiratory syncytial virus (RSV) or a fragment, variant or derivativethereof.

For the sake of clarity and readability the following scientificbackground information and definitions are provided. Any technicalfeatures disclosed thereby can be part of each and every embodiment ofthe invention. Additional definitions and explanations can be providedin the context of this disclosure.

Immune system: The immune system may protect organisms from infection.If a pathogen breaks through a physical barrier of an organism andenters this organism, the innate immune system provides an immediate,but non-specific response. If pathogens evade this innate response,vertebrates possess a second layer of protection, the adaptive immunesystem. Here, the immune system adapts its response during an infectionto improve its recognition of the pathogen. This improved response isthen retained after the pathogen has been eliminated, in the form of animmunological memory, and allows the adaptive immune system to mountfaster and stronger attacks each time this pathogen is encountered.According to this, the immune system comprises the innate and theadaptive immune system. Each of these two parts contains so calledhumoral and cellular components.

Immune response: An immune response may typically either be a specificreaction of the adaptive immune system to a particular antigen (socalled specific or adaptive immune response) or an unspecific reactionof the innate immune system (so called unspecific or innate immuneresponse). The invention relates to the core to specific reactions(adaptive immune responses) of the adaptive immune system. Particularly,it relates to adaptive immune responses to infections by viruses likee.g. RSV infections. However, this specific response can be supported byan additional unspecific reaction (innate immune response). Therefore,the invention also relates to a compound for simultaneous stimulation ofthe innate and the adaptive immune system to evoke an efficient adaptiveimmune response.

Adaptive immune system: The adaptive immune system is composed of highlyspecialized, systemic cells and processes that eliminate or preventpathogenic growth. The adaptive immune response provides the vertebrateimmune system with the ability to recognize and remember specificpathogens (to generate immunity), and to mount stronger attacks eachtime the pathogen is encountered. The system is highly adaptable becauseof somatic hypermutation (a process of increased frequency of somaticmutations), and V(D)J recombination (an irreversible geneticrecombination of antigen receptor gene segments). This mechanism allowsa small number of genes to generate a vast number of different antigenreceptors, which are then uniquely expressed on each individuallymphocyte. Because the gene rearrangement leads to an irreversiblechange in the DNA of each cell, all of the progeny (offspring) of thatcell will then inherit genes encoding the same receptor specificity,including the Memory B cells and Memory T cells that are the keys tolong-lived specific immunity. Immune network theory is a theory of howthe adaptive immune system works, that is based on interactions betweenthe variable regions of the receptors of T cells, B cells and ofmolecules made by T cells and B cells that have variable regions.

Adaptive immune response: The adaptive immune response is typicallyunderstood to be antigen-specific. Antigen specificity allows for thegeneration of responses that are tailored to specific antigens,pathogens or pathogen-infected cells. The ability to mount thesetailored responses is maintained in the body by “memory cells”. Should apathogen infect the body more than once, these specific memory cells areused to quickly eliminate it. In this context, the first step of anadaptive immune response is the activation of naïve antigen-specific Tcells or different immune cells able to induce an antigen-specificimmune response by antigen-presenting cells. This occurs in the lymphoidtissues and organs through which naïve T cells are constantly passing.Cell types that can serve as antigen-presenting cells are inter aliadendritic cells, macrophages, and B cells. Each of these cells has adistinct function in eliciting immune responses. Dendritic cells take upantigens by phagocytosis and macropinocytosis and are stimulated bycontact with e.g. a foreign antigen to migrate to the local lymphoidtissue, where they differentiate into mature dendritic cells.Macrophages ingest particulate antigens such as bacteria and are inducedby infectious agents or other appropriate stimuli to express MHCmolecules. The unique ability of B cells to bind and internalize solubleprotein antigens via their receptors may also be important to induce Tcells. Presenting the antigen on MHC molecules leads to activation of Tcells which induces their proliferation and differentiation into armedeffector T cells. The most important function of effector T cells is thekilling of infected cells by CD8+ cytotoxic T cells and the activationof macrophages by Th1 cells which together make up cell-mediatedimmunity, and the activation of B cells by both Th2 and Th1 cells toproduce different classes of antibody, thus driving the humoral immuneresponse. T cells recognize an antigen by their T cell receptors whichdo not recognize and bind antigen directly, but instead recognize shortpeptide fragments e.g. of pathogen-derived protein antigens, which arebound to MHC molecules on the surfaces of other cells.

Cellular immunity/cellular immune response: Cellular immunity relatestypically to the activation of macrophages, natural killer cells (NK),antigen-specific cytotoxic T-lymphocytes, and the release of variouscytokines in response to an antigen. In a more general way, cellularimmunity is not related to antibodies but to the activation of cells ofthe immune system. A cellular immune response is characterized e.g. byactivating antigen-specific cytotoxic T-lymphocytes that are able toinduce apoptosis in body cells displaying epitopes of an antigen ontheir surface, such as virus-infected cells, cells with intracellularbacteria, and cancer cells displaying tumor antigens; activatingmacrophages and natural killer cells, enabling them to destroypathogens; and stimulating cells to secrete a variety of cytokines thatinfluence the function of other cells involved in adaptive immuneresponses and innate immune responses.

Humoral immunity/humoral immune response: Humoral immunity referstypically to antibody production and the accessory processes that mayaccompany it. A humoral immune response may be typically characterized,e.g., by Th2 activation and cytokine production, germinal centerformation and isotype switching, affinity maturation and memory cellgeneration. Humoral immunity also typically may refer to the effectorfunctions of antibodies, which include pathogen and toxinneutralization, classical complement activation, and opsonin promotionof phagocytosis and pathogen elimination.

Innate immune system: The innate immune system, also known asnon-specific immune system, comprises the cells and mechanisms thatdefend the host from infection by other organisms in a non-specificmanner. This means that the cells of the innate system recognize andrespond to pathogens in a generic way, but unlike the adaptive immunesystem, it does not confer long-lasting or protective immunity to thehost. The innate immune system may be e.g. activated by ligands ofpathogen-associated molecular patterns (PAMP) receptors, e.g. Toll-likereceptors (TLRs) or other auxiliary substances such aslipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines,lymphokines, interleukins or chemokines, IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27,IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta,IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors, andhGH, a ligand of human Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, TLR10, a ligand of murine Toll-like receptorTLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11,TLR12 or TLR13, a ligand of a NOD-like receptor, a ligand of a RIG-Ilike receptor, an immunostimulatory nucleic acid, an immunostimulatoryRNA (isRNA), a CpG-DNA, an antibacterial agent, or an anti-viral agent.Typically a response of the innate immune system includes recruitingimmune cells to sites of infection, through the production of chemicalfactors, including specialized chemical mediators, called cytokines;activation of the complement cascade; identification and removal offoreign substances present in organs, tissues, the blood and lymph, byspecialized white blood cells; activation of the adaptive immune systemthrough a process known as antigen presentation; and/or acting as aphysical and chemical barrier to infectious agents.

Adjuvant/adjuvant component: An adjuvant or an adjuvant component in thebroadest sense is typically a (e.g. pharmacological or immunological)agent or composition that may modify, e.g. enhance, the efficacy ofother agents, such as a drug or vaccine. Conventionally the term refersin the context of the invention to a compound or composition that servesas a carrier or auxiliary substance for immunogens and/or otherpharmaceutically active compounds. It is to be interpreted in a broadsense and refers to a broad spectrum of substances that are able toincrease the immunogenicity of antigens incorporated into orco-administered with an adjuvant in question. In the context of thepresent invention an adjuvant will preferably enhance the specificimmunogenic effect of the active agents of the present invention.Typically, “adjuvant” or “adjuvant component” has the same meaning andcan be used mutually. Adjuvants may be divided, e.g., into immunopotentiators, antigenic delivery systems or even combinations thereof.

The term “adjuvant” is typically understood not to comprise agents whichconfer immunity by themselves. An adjuvant assists the immune systemunspecifically to enhance the antigen-specific immune response by e.g.promoting presentation of an antigen to the immune system or inductionof an unspecific innate immune response. Furthermore, an adjuvant maypreferably e.g. modulate the antigen-specific immune response by e.g.shifting the dominating Th2-based antigen specific response to a moreTh1-based antigen specific response or vice versa. Accordingly, anadjuvant may favourably modulate cytokine expression/secretion, antigenpresentation, type of immune response etc.

Immunostimulatory RNA: An immunostimulatory RNA (isRNA) in the contextof the invention may typically be a RNA that is able to induce an innateimmune response itself. It usually does not have an open reading frameand thus does not provide a peptide-antigen or immunogen but elicits aninnate immune response e.g. by binding to a specific kind ofToll-like-receptor (TLR) or other suitable receptors. However, of coursealso mRNAs having an open reading frame and coding for a peptide/protein(e.g. an antigenic function) may induce an innate immune response.

Antigen: According to the present invention, the term “antigen” referstypically to a substance which may be recognized by the immune systemand may be capable of triggering an antigen-specific immune response,e.g. by formation of antibodies or antigen-specific T-cells as part ofan adaptive immune response. An antigen may be a protein or peptide. Inthis context, the first step of an adaptive immune response is theactivation of naïve antigen-specific T cells by antigen-presentingcells. This occurs in the lymphoid tissues and organs through whichnaïve T cells are constantly passing. The three cell types that canserve as antigen-presenting cells are dendritic cells, macrophages, andB cells. Each of these cells has a distinct function in eliciting immuneresponses. Tissue dendritic cells take up antigens by phagocytosis andmacropinocytosis and are stimulated by infection to migrate to the locallymphoid tissue, where they differentiate into mature dendritic cells.Macrophages ingest particulate antigens such as bacteria and are inducedby infectious agents to express MHC class II molecules. The uniqueability of B cells to bind and internalize soluble protein antigens viatheir receptors may be important to induce T cells. By presenting theantigen on MHC molecules leads to activation of T cells which inducestheir proliferation and differentiation into armed effector T cells. Themost important function of effector T cells is the killing of infectedcells by CD8+ cytotoxic T cells and the activation of macrophages by TH1cells which together make up cell-mediated immunity, and the activationof B cells by both TH2 and TH1 cells to produce different classes ofantibody, thus driving the humoral immune response. T cells recognize anantigen by their T cell receptors which does not recognize and bindantigen directly, but instead recognize short peptide fragments e.g. ofpathogens' protein antigens, which are bound to MHC molecules on thesurfaces of other cells. T cells fall into two major classes that havedifferent effector functions. The two classes are distinguished by theexpression of the cell-surface proteins CD4 and CD8. These two types ofT cells differ in the class of MHC molecule that they recognize. Thereare two classes of MHC molecules—MHC class I and MHC class IImolecules—which differ in their structure and expression pattern ontissues of the body. CD4⁺ T cells bind to a MHC class II molecule andCD8⁺ T cells to a MHC class I molecule. MHC class I and MHC class IImolecules have distinct distributions among cells that reflect thedifferent effector functions of the T cells that recognize them. MHCclass I molecules present peptides of cytosolic and nuclear origin e.g.from pathogens, commonly viruses, to CD8⁺ T cells, which differentiateinto cytotoxic T cells that are specialized to kill any cell that theyspecifically recognize. Almost all cells express MHC class I molecules,although the level of constitutive expression varies from one cell typeto the next. But not only pathogenic peptides from viruses are presentedby MHC class I molecules, also self-antigens like tumour antigens arepresented by them. MHC class I molecules bind peptides from proteinsdegraded in the cytosol and transported in the endoplasmic reticulum.The CD8⁺ T cells that recognize MHC class I:peptide complexes at thesurface of infected cells are specialized to kill any cells displayingforeign peptides and so rid the body of cells infected with viruses andother cytosolic pathogens. The main function of CD4⁺ T cells (CD4⁺helper T cells) that recognize MHC class II molecules is to activateother effector cells of the immune system. Thus MHC class II moleculesare normally found on B lymphocytes, dendritic cells, and macrophages,cells that participate in immune responses, but not on other tissuecells. Macrophages, for example, are activated to kill theintravesicular pathogens they harbour, and B cells to secreteimmunoglobulins against foreign molecules. MHC class II molecules areprevented from binding to peptides in the endoplasmic reticulum and thusMHC class II molecules bind peptides from proteins which are degraded inendosomes. They can capture peptides from pathogens that have enteredthe vesicular system of macrophages, or from antigens internalized byimmature dendritic cells or the immunoglobulin receptors of B cells.Pathogens that accumulate in large numbers inside macrophage anddendritic cell vesicles tend to stimulate the differentiation of TH1cells, whereas extracellular antigens tend to stimulate the productionof TH2 cells. TH1 cells activate the microbicidal properties ofmacrophages and induce B cells to make IgG antibodies that are veryeffective of opsonising extracellular pathogens for ingestion byphagocytic cells, whereas TH2 cells initiate the humoral response byactivating naïve B cells to secrete IgM, and induce the production ofweakly opsonising antibodies such as IgG1 and IgG3 (mouse) and IgG2 andIgG4 (human) as well as IgA and IgE (mouse and human).

Epitope (also called “antigen determinant”): T cell epitopes or parts ofthe proteins in the context of the present invention may comprisefragments preferably having a length of about 6 to about 20 or even moreamino acids, e.g. fragments as processed and presented by MHC class Imolecules, preferably having a length of about 8 to about 10 aminoacids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), or fragmentsas processed and presented by MHC class II molecules, preferably havinga length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18,19, 20 or even more amino acids, wherein these fragments may be selectedfrom any part of the amino acid sequence. These fragments are typicallyrecognized by T cells in form of a complex consisting of the peptidefragment and an MHC molecule.

B cell epitopes are typically fragments located on the outer surface of(native) protein or peptide antigens as defined herein, preferablyhaving 5 to 15 amino acids, more preferably having 5 to 12 amino acids,even more preferably having 6 to 9 amino acids, which may be recognizedby antibodies, i.e. in their native form.

Such epitopes of proteins or peptides may furthermore be selected fromany of the herein mentioned variants of such proteins or peptides. Inthis context antigenic determinants can be conformational ordiscontinuous epitopes which are composed of segments of the proteins orpeptides as defined herein that are discontinuous in the amino acidsequence of the proteins or peptides as defined herein but are broughttogether in the three-dimensional structure or continuous or linearepitopes which are composed of a single polypeptide chain.

Vaccine: A vaccine is typically understood to be a prophylactic ortherapeutic material providing at least one antigen or antigenicfunction. The antigen or antigenic function may stimulate the body'sadaptive immune system to provide an adaptive immune response.

Antigen-providing mRNA: An antigen-providing mRNA in the context of theinvention may typically be an mRNA, having at least one open readingframe that can be translated by a cell or an organism provided with thatmRNA. The product of this translation is a peptide or protein that mayact as an antigen, preferably as an immunogen. The product may also be afusion protein composed of more than one immunogen, e.g. a fusionprotein that consist of two or more epitopes, peptides or proteinsderived from the same or different virus-proteins, wherein the epitopes,peptides or proteins may be linked by linker sequences.

Bi-/multicistronic mRNA: mRNA, that typically may have two (bicistronic)or more (multicistronic) open reading frames (ORF). An open readingframe in this context is a sequence of several nucleotide triplets(codons) that can be translated into a peptide or protein. Translationof such a mRNA yields two (bicistronic) or more (multicistronic)distinct translation products (provided the ORFs are not identical). Forexpression in eukaryotes such mRNAs may for example comprise an internalribosomal entry site (IRES) sequence.

A 5′-CAP is typically a modified nucleotide, particularly a guaninenucleotide, added to the 5′ end of an mRNA-molecule. Preferably, the5′-CAP is added using a 5′-5′-triphosphate linkage (also named m7GpppN).Further examples of 5′-CAP structures include glyceryl, inverted deoxyabasic residue (moiety), 4′,5′ methylene nucleotide,1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides,alpha-nucleotide, modified base nucleotide, threo-pentofuranosylnucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutylnucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3′-3′-invertednucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-invertednucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediolphosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate,3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging ornon-bridging methylphosphonate moiety. These modified 5′-CAP structuresmay be used in the context of the present invention to modify theinventive mRNA sequence. Further modified 5′-CAP structures which may beused in the context of the present invention are CAP1 (methylation ofthe ribose of the adjacent nucleotide of m7GpppN), CAP2 (methylation ofthe ribose of the 2^(nd) nucleotide downstream of the m7GpppN), CAP3(methylation of the ribose of the 3^(rd) nucleotide downstream of them7GpppN), CAP4 (methylation of the ribose of the 4^(th) nucleotidedownstream of the m7GpppN), ARCA (anti-reverse CAP analogue, modifiedARCA (e.g. phosphothioate modified ARCA), inosine, N1-methyl-guanosine,2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

Fragments of proteins: “Fragments” of proteins or peptides in thecontext of the present invention may, typically, comprise a sequence ofa protein or peptide as defined herein, which is, with regard to itsamino acid sequence (or its encoded nucleic acid molecule), N-terminallyand/or C-terminally truncated compared to the amino acid sequence of theoriginal (native) protein (or its encoded nucleic acid molecule). Suchtruncation may thus occur either on the amino acid level orcorrespondingly on the nucleic acid level. A sequence identity withrespect to such a fragment as defined herein may therefore preferablyrefer to the entire protein or peptide as defined herein or to theentire (coding) nucleic acid molecule of such a protein or peptide.

Fragments of proteins or peptides in the context of the presentinvention may furthermore comprise a sequence of a protein or peptide asdefined herein, which has a length of for example at least 5 aminoacids, preferably a length of at least 6 amino acids, preferably atleast 7 amino acids, more preferably at least 8 amino acids, even morepreferably at least 9 amino acids; even more preferably at least 10amino acids; even more preferably at least 11 amino acids; even morepreferably at least 12 amino acids; even more preferably at least 13amino acids; even more preferably at least 14 amino acids; even morepreferably at least 15 amino acids; even more preferably at least 16amino acids; even more preferably at least 17 amino acids; even morepreferably at least 18 amino acids; even more preferably at least 19amino acids; even more preferably at least 20 amino acids; even morepreferably at least 25 amino acids; even more preferably at least 30amino acids; even more preferably at least 35 amino acids; even morepreferably at least 50 amino acids; or most preferably at least 100amino acids. For example such fragment may have a length of about 6 toabout 20 or even more amino acids, e.g. fragments as processed andpresented by MHC class I molecules, preferably having a length of about8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 6, 7, 11, or 12amino acids), or fragments as processed and presented by MHC class IImolecules, preferably having a length of about 13 or more amino acids,e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, whereinthese fragments may be selected from any part of the amino acidsequence. These fragments are typically recognized by T-cells in form ofa complex consisting of the peptide fragment and an MHC molecule, i.e.the fragments are typically not recognized in their native form.Fragments of proteins or peptides may comprise at least one epitope ofthose proteins or peptides. Furthermore also domains of a protein, likethe extracellular domain, the intracellular domain or the transmembranedomain and shortened or truncated versions of a protein may beunderstood to comprise a fragment of a protein.

Variants of proteins: “Variants” of proteins or peptides as defined inthe context of the present invention may be generated, having an aminoacid sequence which differs from the original sequence in one or moremutation(s), such as one or more substituted, inserted and/or deletedamino acid(s). Preferably, these fragments and/or variants have the samebiological function or specific activity compared to the full-lengthnative protein, e.g. its specific antigenic property. “Variants” ofproteins or peptides as defined in the context of the present inventionmay comprise conservative amino acid substitution(s) compared to theirnative, i.e. non-mutated physiological, sequence. Those amino acidsequences as well as their encoding nucleotide sequences in particularfall under the term variants as defined herein. Substitutions in whichamino acids, which originate from the same class, are exchanged for oneanother are called conservative substitutions. In particular, these areamino acids having aliphatic side chains, positively or negativelycharged side chains, aromatic groups in the side chains or amino acids,the side chains of which can enter into hydrogen bridges, e.g. sidechains which have a hydroxyl function. This means that e.g. an aminoacid having a polar side chain is replaced by another amino acid havinga likewise polar side chain, or, for example, an amino acidcharacterized by a hydrophobic side chain is substituted by anotheramino acid having a likewise hydrophobic side chain (e.g. serine(threonine) by threonine (serine) or leucine (isoleucine) by isoleucine(leucine)). Insertions and substitutions are possible, in particular, atthose sequence positions which cause no modification to thethree-dimensional structure or do not affect the binding region.Modifications to a three-dimensional structure by insertion(s) ordeletion(s) can easily be determined e.g. using CD spectra (circulardichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORDof Polypeptides, in: Modern Physical Methods in Biochemistry, Neubergeret al. (ed.), Elsevier, Amsterdam).

A “variant” of a protein or peptide may have at least 70%, 75%, 80%,85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of 10, 20,30, 50, 75 or 100 amino acids of such protein or peptide.

Furthermore, variants of proteins or peptides as defined herein, whichmay be encoded by a nucleic acid molecule, may also comprise thosesequences, wherein nucleotides of the encoding nucleic acid sequence areexchanged according to the degeneration of the genetic code, withoutleading to an alteration of the respective amino acid sequence of theprotein or peptide, i.e. the amino acid sequence or at least partthereof may not differ from the original sequence in one or moremutation(s) within the above meaning.

Identity of a sequence: In order to determine the percentage to whichtwo sequences are identical, e.g. nucleic acid sequences or amino acidsequences as defined herein, preferably the amino acid sequences encodedby a nucleic acid sequence of the polymeric carrier as defined herein orthe amino acid sequences themselves, the sequences can be aligned inorder to be subsequently compared to one another. Therefore, e.g. aposition of a first sequence may be compared with the correspondingposition of the second sequence. If a position in the first sequence isoccupied by the same component (residue) as is the case at a position inthe second sequence, the two sequences are identical at this position.If this is not the case, the sequences differ at this position. Ifinsertions occur in the second sequence in comparison to the firstsequence, gaps can be inserted into the first sequence to allow afurther alignment. If deletions occur in the second sequence incomparison to the first sequence, gaps can be inserted into the secondsequence to allow a further alignment. The percentage to which twosequences are identical is then a function of the number of identicalpositions divided by the total number of positions including thosepositions which are only occupied in one sequence. The percentage towhich two sequences are identical can be determined using a mathematicalalgorithm. A preferred, but not limiting, example of a mathematicalalgorithm which can be used is the algorithm of Karlin et al. (1993),PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res.,25:3389-3402. Such an algorithm is integrated in the BLAST program.Sequences which are identical to the sequences of the present inventionto a certain extent can be identified by this program.

Derivative of a protein or peptide: A derivative of a peptide or proteinis typically understood to be a molecule that is derived from anothermolecule, such as said peptide or protein. A “derivative” of a peptideor protein also encompasses fusions comprising a peptide or protein usedin the present invention. For example, the fusion comprises a label,such as, for example, an epitope, e.g., a FLAG epitope or a V5 epitope.For example, the epitope is a FLAG epitope. Such a tag is useful for,for example, purifying the fusion protein.

Monocistronic mRNA: A monocistronic mRNA may typically be an mRNA, thatencodes only one open reading frame. An open reading frame in thiscontext is a sequence of several nucleotide triplets (codons) that canbe translated into a peptide or protein.

Nucleic acid: The term nucleic acid means any DNA- or RNA-molecule andis used synonymous with polynucleotide. Wherever herein reference ismade to a nucleic acid or nucleic acid sequence encoding a particularprotein and/or peptide, said nucleic acid or nucleic acid sequence,respectively, preferably also comprises regulatory sequences allowing ina suitable host, e.g. a human being, its expression, i.e. transcriptionand/or translation of the nucleic acid sequence encoding the particularprotein or peptide.

Peptide: A peptide is a polymer of amino acid monomers. Usually themonomers are linked by peptide bonds. The term “peptide” does not limitthe length of the polymer chain of amino acids. In some embodiments ofthe present invention a peptide may for example contain less than 50monomer units. Longer peptides are also called polypeptides, typicallyhaving 50 to 600 monomeric units, more specifically 50 to 300 monomericunits.

Pharmaceutically effective amount: A pharmaceutically effective amountin the context of the invention is typically understood to be an amountthat is sufficient to induce an immune response.

Protein: A protein typically consists of one or more peptides and/orpolypeptides folded into 3-dimensional form, facilitating a biologicalfunction.

Poly (C) sequence: A poly-(C)-sequence is typically a long sequence ofcytosine nucleotides, typically about 10 to about 200 cytosinenucleotides, preferably about 10 to about 100 cytosine nucleotides, morepreferably about 10 to about 70 cytosine nucleotides or even morepreferably about 20 to about 50 or even about 20 to about 30 cytosinenucleotides. A poly(C) sequence may preferably be located 3′ of thecoding region comprised by a nucleic acid.

Poly-A-tail: A poly-A-tail also called “3′-poly(A) tail” is typically along sequence of adenosine nucleotides of up to about 400 adenosinenucleotides, e.g. from about 25 to about 400, preferably from about 50to about 400, more preferably from about 50 to about 300, even morepreferably from about 50 to about 250, most preferably from about 60 toabout 250 adenosine nucleotides, added to the 3′ end of a RNA.

Stabilized nucleic acid: A stabilized nucleic acid, typically, exhibitsa modification increasing resistance to in vivo degradation (e.g.degradation by an exo- or endo-nuclease) and/or ex vivo degradation(e.g. by the manufacturing process prior to vaccine administration, e.g.in the course of the preparation of the vaccine solution to beadministered). Stabilization of RNA can, e.g., be achieved by providinga 5′-CAP-Structure, a Poly-A-Tail, or any other UTR-modification. It canalso be achieved by backbone-modification or modification of theG/C-content of the nucleic acid. Various other methods are known in theart and conceivable in the context of the invention.

Carrier/polymeric carrier: A carrier in the context of the invention maytypically be a compound that facilitates transport and/or complexationof another compound. Said carrier may form a complex with said othercompound. A polymeric carrier is a carrier that is formed of a polymer.

Cationic component: The term “cationic component” typically refers to acharged molecule, which is positively charged (cation) at a pH value oftypically about 1 to 9, preferably of a pH value of or below 9 (e.g. 5to 9), of or below 8 (e.g. 5 to 8), of or below 7 (e.g. 5 to 7), mostpreferably at physiological pH values, e.g. about 7.3 to 7.4.Accordingly, a cationic peptide, protein or polymer according to thepresent invention is positively charged under physiological conditions,particularly under physiological salt conditions of the cell in vivo. Acationic peptide or protein preferably contains a larger number ofcationic amino acids, e.g. a larger number of Arg, His, Lys or Orn thanother amino acid residues (in particular more cationic amino acids thananionic amino acid residues like Asp or Glu) or contains blockspredominantly formed by cationic amino acid residues. The definition“cationic” may also refer to “polycationic” components.

Vehicle: An agent, e.g. a carrier, that may typically be used within apharmaceutical composition or vaccine for facilitating administering ofthe components of the pharmaceutical composition or vaccine to anindividual.

3′-untranslated region (3′UTR): A 3′UTR is typically the part of an mRNAwhich is located between the protein coding region (i.e. the openreading frame) and the poly(A) sequence of the mRNA. A 3′UTR of the mRNAis not translated into an amino acid sequence. The 3′UTR sequence isgenerally encoded by the gene which is transcribed into the respectivemRNA during the gene expression process. The genomic sequence is firsttranscribed into pre-mature mRNA, which comprises optional introns. Thepre-mature mRNA is then further processed into mature mRNA in amaturation process. This maturation process comprises the steps of5′-Capping, splicing the pre-mature mRNA to excise optional introns andmodifications of the 3′-end, such as polyadenylation of the 3′-end ofthe pre-mature mRNA and optional endo- or exonuclease cleavages etc. Inthe context of the present invention, a 3′UTR corresponds to thesequence of a mature mRNA which is located 3′ to the stop codon of theprotein coding region, preferably immediately 3′ to the stop codon ofthe protein coding region, and which extends to the 5′-side of thepoly(A) sequence, preferably to the nucleotide immediately 5′ to thepoly(A) sequence. The term “corresponds to” means that the 3′UTRsequence may be an RNA sequence, such as in the mRNA sequence used fordefining the 3′UTR sequence, or a DNA sequence which corresponds to suchRNA sequence. In the context of the present invention, the term “a 3′UTRof a gene”, such as “a 3′UTR of an albumin gene”, is the sequence whichcorresponds to the 3′UTR of the mature mRNA derived from this gene, i.e.the mRNA obtained by transcription of the gene and maturation of thepre-mature mRNA. The term “3′UTR of a gene” encompasses the DNA sequenceand the RNA sequence of the 3′UTR.

5′-untranslated region (5′UTR): A 5′-UTR is typically understood to be aparticular section of messenger RNA (mRNA). It is located 5′ of the openreading frame of the mRNA. Typically, the 5′UTR starts with thetranscriptional start site and ends one nucleotide before the startcodon of the open reading frame. The 5′-UTR may comprise elements forcontrolling gene expression, also called regulatory elements. Suchregulatory elements may be, for example, ribosomal binding sites or a5′-Terminal Oligopyrimidine Tract. The 5′UTR may beposttranscriptionally modified, for example by addition of a 5′-CAP. Inthe context of the present invention, a 5′UTR corresponds to thesequence of a mature mRNA which is located between the 5′-CAP and thestart codon. Preferably, the 5′UTR corresponds to the sequence whichextends from a nucleotide located 3′ to the 5′-CAP, preferably from thenucleotide located immediately 3′ to the 5′-CAP, to a nucleotide located5′ to the start codon of the protein coding region, preferably to thenucleotide located immediately 5′ to the start codon of the proteincoding region. The nucleotide located immediately 3′ to the 5′-CAP of amature mRNA typically corresponds to the transcriptional start site. Theterm “corresponds to” means that the 5′UTR sequence may be an RNAsequence, such as in the mRNA sequence used for defining the 5′UTRsequence, or a DNA sequence which corresponds to such RNA sequence. Inthe context of the present invention, the term “a 5′UTR of a gene”, suchas “a 5′UTR of a TOP gene”, is the sequence which corresponds to the5′UTR of the mature mRNA derived from this gene, i.e. the mRNA obtainedby transcription of the gene and maturation of the pre-mature mRNA. Theterm “5′UTR of a gene” encompasses the DNA sequence and the RNA sequenceof the 5′UTR.

5′Terminal Oligopyrimidine Tract (TOP): The 5′terminal oligopyrimidinetract (TOP) is typically a stretch of pyrimidine nucleotides located atthe 5′ terminal region of a nucleic acid molecule, such as the 5′terminal region of certain mRNA molecules or the 5′ terminal region of afunctional entity, e.g. the transcribed region, of certain genes. Thesequence starts with a cytidine, which usually corresponds to thetranscriptional start site, and is followed by a stretch of usuallyabout 3 to 30 pyrimidine nucleotides. For example, the TOP may comprise3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or even more nucleotides. The pyrimidinestretch and thus the 5′ TOP ends one nucleotide 5′ to the first purinenucleotide located downstream of the TOP. Messenger RNA that contains a5′terminal oligopyrimidine tract is often referred to as TOP mRNA.Accordingly, genes that provide such messenger RNAs are referred to asTOP genes. TOP sequences have, for example, been found in genes andmRNAs encoding peptide elongation factors and ribosomal proteins.

TOP motif: In the context of the present invention, a TOP motif is anucleic acid sequence which corresponds to a 5′TOP as defined above.Thus, a TOP motif in the context of the present invention is preferablya stretch of pyrimidine nucleotides having a length of 3-30 nucleotides.Preferably, the TOP-motif consists of at least 3 pyrimidine nucleotides,preferably at least 4 pyrimidine nucleotides, preferably at least 5pyrimidine nucleotides, more preferably at least 6 nucleotides, morepreferably at least 7 nucleotides, most preferably at least 8 pyrimidinenucleotides, wherein the stretch of pyrimidine nucleotides preferablystarts at its 5′ end with a cytosine nucleotide. In TOP genes and TOPmRNAs, the TOP-motif preferably starts at its 5′end with thetranscriptional start site and ends one nucleotide 5′ to the first purinresidue in said gene or mRNA. A TOP motif in the sense of the presentinvention is preferably located at the 5′end of a sequence whichrepresents a 5′UTR or at the 5′end of a sequence which codes for a5′UTR. Thus, preferably, a stretch of 3 or more pyrimidine nucleotidesis called “TOP motif” in the sense of the present invention if thisstretch is located at the 5′end of a respective sequence, such as theinventive mRNA, the 5′UTR element of the inventive mRNA, or the nucleicacid sequence which is derived from the 5′UTR of a TOP gene as describedherein. In other words, a stretch of 3 or more pyrimidine nucleotideswhich is not located at the 5′-end of a 5′UTR or a 5′UTR element butanywhere within a 5′UTR or a 5′UTR element is preferably not referred toas “TOP motif”.

TOP gene: TOP genes are typically characterised by the presence of a 5′terminal oligopyrimidine tract. Furthermore, most TOP genes arecharacterized by a growth-associated translational regulation. However,also TOP genes with a tissue specific translational regulation areknown. As defined above, the 5′UTR of a TOP gene corresponds to thesequence of a 5′UTR of a mature mRNA derived from a TOP gene, whichpreferably extends from the nucleotide located 3′ to the 5′-CAP to thenucleotide located 5′ to the start codon. A 5′UTR of a TOP genetypically does not comprise any start codons, preferably no upstreamAUGs (uAUGs) or upstream open reading frames (uORFs). Therein, upstreamAUGs and upstream open reading frames are typically understood to beAUGs and open reading frames that occur 5′ of the start codon (AUG) ofthe open reading frame that should be translated. The 5′UTRs of TOPgenes are generally rather short. The lengths of 5′UTRs of TOP genes mayvary between 20 nucleotides up to 500 nucleotides, and are typicallyless than about 200 nucleotides, preferably less than about 150nucleotides, more preferably less than about 100 nucleotides. Exemplary5′UTRs of TOP genes in the sense of the present invention are thenucleic acid sequences extending from the nucleotide at position 5 tothe nucleotide located immediately 5′ to the start codon (e.g. the ATG)in the sequences according to SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQID NO. 1421 and SEQ ID NO. 14221-1363 of the patent applicationPCT/EP2012/002448WO2013/143700 or homologs or variants thereof, whosedisclosure is incorporated herewith by reference. In this context aparticularly preferred fragment of a 5′UTR of a TOP gene is a 5′UTR of aTOP gene lacking the 5′TOP motif. The term ‘5′UTR of a TOP gene’preferably refers to the 5′UTR of a naturally occurring TOP gene.

Fragment of a nucleic acid sequence, particularly an mRNA: A fragment ofa nucleic acid sequence consists of a continuous stretch of nucleotidescorresponding to a continuous stretch of nucleotides in the full-lengthnucleic acid sequence which is the basis for the nucleic acid sequenceof the fragment, which represents at least 20%, preferably at least 30%,more preferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, even more preferably at least 70%, even morepreferably at least 80%, and most preferably at least 90% of thefull-length nucleic acid sequence. Such a fragment, in the sense of thepresent invention, is preferably a functional fragment of thefull-length nucleic acid sequence.

Variant of a nucleic acid sequence, particularly an mRNA: A variant of anucleic acid sequence refers to a variant of nucleic acid sequenceswhich forms the basis of a nucleic acid sequence. For example, a variantnucleic acid sequence may exhibit one or more nucleotide deletions,insertions, additions and/or substitutions compared to the nucleic acidsequence from which the variant is derived. Preferably, a variant of anucleic acid sequence is at least 40%, preferably at least 50%, morepreferably at least 60%, more preferably at least 70%, even morepreferably at least 80%, even more preferably at least 90%, mostpreferably at least 95% identical to the nucleic acid sequence thevariant is derived from. Preferably, the variant is a functionalvariant. A “variant” of a nucleic acid sequence may have at least 70%,75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a stretchof 10, 20, 30, 50, 75 or 100 nucleotide of such nucleic acid sequence.

Homolog of a nucleic acid sequence: The term “homolog” of a nucleic acidsequence refers to sequences of other species than the particularsequence. It is particular preferred that the nucleic acid sequence isof human origin and therefore it is preferred that the homolog is ahomolog of a human nucleic acid sequence.

Jet injection: The term “jet injection”, as used herein, refers to aneedle-free injection method, wherein a fluid containing at least oneinventive mRNA sequence and, optionally, further suitable excipients isforced through an orifice, thus generating an ultra-fine liquid streamof high pressure that is capable of penetrating mammalian skin and,depending on the injection settings, subcutaneous tissue or muscletissue. In principle, the liquid stream forms a hole in the skin,through which the liquid stream is pushed into the target tissue.Preferably, jet injection is used for intradermal, subcutaneous orintramuscular injection of the mRNA sequence according to the invention.In a preferred embodiment, jet injection is used for intramuscularinjection of the inventive mRNA. In a further preferred embodiment, jetinjection is used for intradermal injection of the inventive mRNa.

The present invention is based on the surprising finding of the presentinventors that an mRNA sequence comprising a coding region, encoding atleast one antigenic peptide or protein of Respiratory syncytial virus(RSV) induces antigen-specific immune responses and therefore prevent orat least minimize Respiratory syncytial virus (RSV) infections. It wasvery surprising for the inventors that the inventive mRNA sequenceinduces at least the same immune responses than vaccines based oninactivated RSV which consists of the whole virus. Even moresurprisingly the inventive mRNA sequence coding for an antigenic proteinof RSV induced antigen-specific CD8+-T cells in contrast to a vaccinebased on an inactivated RSV. Additionally, in a cotton rat RSV challengemodel, the virus titers in the nose and in the lung of mRNA vaccinatedanimals were much lower compared to animals vaccinated with vaccinesbased on an inactivated RSV virus. With regard to safety the inventorscould show that the mRNA-based RSV vaccine showed no hints forvaccine-mediated disease enhancement, in terms of lung pathology,compared to a vaccine based on formalin-inactivated virus. Furthermore,it has surprisingly been found by the inventors that already one singlevaccination with the inventive mRNA sequence was sufficient foreliciting an immune response against the administered antigen(s).Specifically, it has been found that one single administration,preferably by intradermal or intramuscular injection, of the inventivemRNA is highly efficient in reducing viral titers in the lung afterchallenge with RSV virus.

In summary, the inventive mRNA sequence comprising a coding regionencoding at least one antigenic peptide or protein of Respiratorysyncytial virus (RSV) could provide an effective and safe vaccine,particularly for infants, the elderly and immunocompromised patients.

In this context it is particularly preferred that the inventive mRNAsequence comprises a coding region, encoding at least one antigenicpeptide or protein derived from the fusion protein F, the glycoproteinG, the short hydrophobic protein SH, the matrix protein M, thenucleoprotein N, the large polymerase L, the M2-1 protein, the M2-2protein, the phosphoprotein P, the non-structural protein NS1 or thenon-structural protein NS2 of Respiratory syncytial virus (RSV) or afragment, variant or derivative thereof.

The coding region of the inventive mRNA sequence according to the firstaspect of the present invention may occur as a mono-, bi-, or evenmulticistronic mRNA, i.e. an mRNA sequence which carries the codingsequences of one, two or more proteins or peptides. Such codingsequences in bi-, or even multicistronic mRNAs may be separated by atleast one internal ribosome entry site (IRES) sequence, e.g. asdescribed herein or by signal peptides which induce the cleavage of theresulting polypeptide which comprises several proteins or peptides.

According to the first aspect of the present invention, the inventivemRNA sequence comprises a coding region, encoding at least one antigenicpeptide or protein derived from the fusion protein F, the glycoproteinG, the short hydrophobic protein SH, the matrix protein M, thenucleoprotein N, the large polymerase L, the M2-1 protein, the M2-2protein, the phosphoprotein P, the non-structural protein NS1 or thenon-structural protein NS2 of Respiratory syncytial virus (RSV) or afragment, variant or derivative thereof. In a particularly preferredembodiment of the first aspect of the invention, the inventive mRNAsequence comprises a coding region, encoding at least one antigenicpeptide or protein derived from the fusion protein F, the nucleoproteinN, or the M2-1 protein of Respiratory syncytial virus (RSV) or afragment, variant or derivative thereof.

In this context, the amino acid sequence of the at least one antigenicpeptide or protein may be selected from any peptide or protein derivedfrom the fusion protein F, the glycoprotein G, the short hydrophobicprotein SH, the matrix protein M, the nucleoprotein N, the largepolymerase L, the M2-1 protein, the M2-2 protein, the phosphoprotein P,the non-structural protein NS1 or the non-structural protein NS2 of anyRSV isolate or from any synthetically engineered RSV peptide or proteinor from a fragment, variant or derivative thereof.

In a particularly preferred embodiment, the full-length protein of thefusion protein F, the glycoprotein G, the short hydrophobic protein SH,the matrix protein M, the nucleoprotein N, the large polymerase L, theM2-1 protein, the M2-2 protein, the phosphoprotein P, the non-structuralprotein NS1 or the non-structural protein NS2 of Respiratory syncytialvirus (RSV) is encoded by the coding region comprised in the inventivemRNA.

In this context, the full-length protein from the fusion protein F andthe nucleoprotein N are particularly preferred. Furthermore a mutant ofthe F protein with a deletion of the cytoplasmic tail is particularlypreferred. An example of such a deletion mutant is the RSV-Fdel 554-574long protein according to (Oomens et al. 2006. J. Virol.80(21):10465-77).

In a further particularly preferred embodiment, a fragment comprising atleast one epitope of the fusion protein F, the glycoprotein G, the shorthydrophobic protein SH, the matrix protein M, the nucleoprotein N, thelarge polymerase L, the M2-1 protein, the M2-2 protein, thephosphoprotein P, the non-structural protein NS1 or the non-structuralprotein NS2 of Respiratory syncytial virus (RSV) is encoded by thecoding region comprised in the inventive mRNA.

Particularly preferred are the amino acid sequences of the RSV strainlong (ATCC VR-26) according to the NCBI accession No. AY911262:

Fusion protein F of the RSV strain ATCC VR-26 long:Amino acid sequence according to SEQ ID No. 1:MELPILKANA ITTILAAVTF CFASSQNITE EFYQSTCSAV SKGYLSALRT GWYTSVITIELSNIKENKCN GTDAKVKLIN QELDKYKNAV TELQLLMQST TAANNRARRELPRFMNYTLN NTKKTNVTLS KKRKRRFLGF LLGVGSAIAS GIAVSKVLHLEGEVNKIKSA LLSTNKAVVS LSNGVSVLTS KVLDLKNYID KQLLPIVNKQSCRISNIETV IEFQQKNNRL LEITREFSVN AGVTTPVSTY MLTNSELLSL INDMPITNDQKKLMSNNVQI VRQQSYSIMS IIKEEVLAYV VQLPLYGVID TPCWKLHTSPLCTTNTKEGS NICLTRTDRG WYCDNAGSVS FFPQAETCKV QSNR VFCDTMNSLTLPSEVN LCNVDIFNPK YDCKIMTSKT DVSSSVITSL GAIVSCYGKTKCTASNKNRG IIKTFSNGCD YVSNKGVDTV SVGNTLYYVN KQEGKSLYVKGEPIINFYDP LVFPSDEFDA SISQVNEKIN QSLAFIRKSD ELLHHVNAGK STTNIMITTIIIVIIVILLS LIAVGLLLYC KARSTPVTLS KDQLSGINNI AFSNGlycoprotein G of the RSV strain ATCC VR-26 long:Amino acid sequence according to SEQ ID No. 2:MSKNKDQRTA KTLEKTWDTL NHLLFISSGL YKLNLKSIAQ ITLSILAMII STSLIITAIIFIASANHKVT LTTAIIQDAT SQIKNTTPTY LTQDPQLGIS FSNLSEITSQ TTTILASTTPGVKSNLQPTT VKTKNTTTTQ TQPSKPTTKQ RQNKPPNKPN NDFHFEVENFVPCSICSNNP TCWAICKRIP NKKPGKKTTT KPTKKPTFKT TKKDLKPQTTKPKEVPTTKP TEEPTINTTK TNITTTLLTN NTTGNPKLTS QMETFHSTSS EGNLSPSQVSTTSEHPSQPS SPPNTTRQShort hydrophobic protein SH of the RSV strain ATCC VR-26 long:Amino acid sequence according to SEQ ID No. 3:MENTSITIEF SSKFWPYFTL IHMITTIISL LIIISIMTAI LNKLCEYNVF HNKTFELPRA RVNTMatrix protein M of the RSV strain ATCC VR-26 long:Amino acid sequence according to SEQ ID No. 4:METYVNKLHE GSTYTAAVQY NVLEKDDDPA SLTIWVPMFQ SSMPADLLIKELANVNILVK QISTPKGPSL RVMINSRSAL LAQMPSKFTI CANVSLDERSKLAYDVTTPC EIKACSLTCL KSKNMLTTVK DLTMKTLNPT HDIIALCEFENIVTSKKVII PTYLRSISVR NKDLNTLENI TTTEFKNAIT NAKIIPYSGL LLVITVTDNKGAFKYIKPQS QFIVDLGAYL EKESIYYVTT NWKHTATRFA IKPMEDNucleoprotein N of the RSV strain ATCC VR-26 long:Amino acid sequence according to SEQ ID No. 5:MALSKVKLND TLNKDQLLSS SKYTIQRSTG DSIDTPNYDV QKHINKLCGM LLITEDANHKFTGLIGMLYA MSRLGREDTI KILRDAGYHV KANGVDVTTH RQDINGKEMK FEVLTLASLTTEIQINIEIE SRKSYKKMLK EMGEVAPEYR HDSPDCGMII LCIAALVITK LAAGDRSGLTAVIRRANNVL KNEMKRYKGL LPKDIANSFY EVFEKHPHFI DVFVHFGIAQ SSTRGGSRVEGIFAGLFMNA YGAGQVMLRW GVLAKSVKNI MLGHASVQAE MEQVVEVYEY AQKLGGEAGFYHILNNPKAS LLSLTQFPHF SSVVLGNAAG LGIMGEYRGT PRNQDLYDAA KAYAEQLKENGVINYSVLDL TAEELEAIKH QLNPKDNDVE LLarge polymerase L of the RSV strain ATCC VR-26 long:Amino acid sequence according to SEQ ID No. 6:MDPIINGNSA NVYLTDSYLK GVISFSECNA LGSYIFNGPY LKNDYTNLIS RONPLIEHMNLKKLNITQSL ISKYHKGEIK LEEPTYFQSL LMTYKSMTSL EQIATTNLLK KIIRRAIEISDVKVYAILNK LGLKEKDKIK SNNGQDEDNS VITTIIKDDI LSAVKDNQSH LKADKNHSTKQKDTIKTTLL KKLMCSMQHP PSWLIHWFNL YTKLNNILTQ YRSNEVKNHG FILIDNQTLSGFQFILNQYG CIVYHKELKR ITVTTYNQFL TWKDISLSRL NVCLITWISN CLNTLNKSLGLRCGFNNVIL TQLFLYGDCI LKLFHNEGFY IIKEVEGFIM SLILNITEED QFRKRFYNSMLNNITDAANK AQKNLLSRVC HTLLDKTVSD NIINGRWIIL LSKFLKLIKL AGDNNLNNLSELYFLFRIFG HPMVDERQAM DAVKVNCNET KFYLLSSLSM LRGAFIYRII KGFVNNYNRWPTLRNAIVLP LRWLTYYKLN TYPSLLELTE RDLIVLSGLR FYREFRLPKK VDLEMIINDKAISPPKNLIW TSFPRNYMPS HIQNYIEHEK LKFSESDKSR RVLEYYLRDN KFNECDLYNCVVNQSYLNNP NHVVSLTGKE RELSVGRMFA MQPGMFRQVQ ILAEKMIAEN ILQFFPESLTRYGDLELQKI LELKAGISNK SNRYNDNYNN YISKCSIITD LSKFNQAFRY ETSCICSDVLDELHGVQSLF SWLHLTIPHV TIICTYRHAP PYIRDHIVDL NNVDEQSGLY RYHMGGIEGWCQKLWTIEAI SLLDLISLKG KESITALING DNQSIDISKP VRLMEGQTHA QADYLLALNSLKLLYKEYAG IGHKLKGTET YISRDMQFMS KTIQHNGVYY PASIKKVLRV GPWINTILDDFKVSLESIGS LTQELEYRGE SLLCSLIFRN VWLYNQIALQ LKNHALCNNK LYLDILKVLKHLKTFFNLDN IDTALTLYMN LPMLFGGGDP NLLYRSFYRR TPDFLTEAIV HSVFILSYYTNHDLKDKLQD LSDDRLNKFL TCIITFDKNP NAEFVTLMRD PQALGSERQA KITSEINRLAVTEVLSTAPN KIFSKSAQHY TTTEIDLNDI MQNIEPTYPH GLRVVYESLP FYKAEKIVNLISGTKSITNI LEKTSAIDLT DIDRATEMMR KNITLLIRIL PLDCNRDKRE ILSMENLSITELSKYVRERS WSLSNIVGVT SPSIMYTMDI KYTTSTIASG IIIEKYNVNS LTRGERGPTKPWVGSSTQEK KTMPVYNRQV LTKKQRDQID LLAKLDWVYA SIDNKDEFME ELSIGTLGLTYEKAKKLFPQ YLSVNYLHRL TVSSRPCEFP ASIPAYRTTN YHFDTSPINR ILTEKYGDEDIDIVFQNCIS FGLSLMSVVE QFTNVCPNRI ILIPKLNEIH LMKPPIFTGD VDIHKLKQVIQKQHMFLPDK ISLTQYVELF LSNKTLKSGS HVNSNLILAH KISDYFHNTY ILSTNLAGHWILIIQLMKDS KGIFEKDWGE GYITDHMFIN LKVFFNAYKT YLLCFHKGYG KAKLECDMNTSDLLCVLELI DSSYWKSMSK VFLEQKVIKY ILSQDASLHR VKGCHSFKLW FLKRLNVAEFTVCPWVVNID YHPTHMKAIL TYIDLVRMGL INIDRIHIKN KHKENDEFYT SNLFYINYNFSDNTHLLTKH IRIANSELEN NYNKLYHPTP ETLENILANP IKSNDKKTLN DYCIGKNVDSIMLPLLSNKK LVKSSAMIRT NYSKQDLYNL FPTVVIDRII DHSGNTAKSN QLYTTTSHQISLVHNSTSLY CMLPWHHINR FNFVFSSTGC KISIEYILKD LKIKDPNCIA FIGEGAGNLLLRTVVELHPD IRYIYRSLKD CNDHSLPIEF LRLYNGHINI DYGENLTIPA TDATNNIHWSYLHIKFAEPI SLFVCDAELP VTVNWSKIII EWSKHVRKCK YCSSVNKCTL IVKYHAQDDIDFKLDNITIL KTYVCLGSKL KGSEVYLVLT IGPANIFPVF NVVQNAKLIL SRTKNFIMPKKADKESIDAN IKSLIPFLCY PITKKGINTA LSKLKSVVSG DILSYSIAGR NEVESNKLINHKHMNILKWF NHVLNFRSTE LNYNHLYMVE STYPYLSELL NSLTTNELKK LIKITGSLLY NFHNEM2-1 protein of the RSV strain ATCC VR-26 long:Amino acid sequence according to SEQ ID No. 7:MSRRNPCKFE IRGHCLNGKR CHESHNYFEW PPHALLVRQN FMLNRILKSM DKSIDTLSEISGAAELDRTE EYALGVVGVL ESYIGSINNI TKQSACVAMS KLLTELNSDD IKKLRDNEELNSPKIRVYNT VISYIESNRK NNKQTIHLLK RLPADVLKKT IKNTLDIHKS ITINNPKELTVSDTNDHAKN NDTT M2-2 protein of the RSV strain ATCC VR-26 long:Amino acid sequence according to SEQ ID No. 8:MTMPKIMILP DKYPCSITSI LITSRCRVTM YNRKNTLYFN QNNPNNHMYS PNQTFNEIHWTSQDLIDTIQ NFLQHLGVIE DIYTIYILVSPhosphoprotein P of the RSV strain ATCC VR-26 long:Amino acid sequence according to SEQ ID No. 9:MEKFAPEFHG EDANNRATKF LESIKGKFTS PKDPKKKDSI ISVNSIDIEV TKESPITSNSTIINPTNETD DNAGNKPNYQ RKPLVSFKED PIPSDNPFSK LYKETIETFD NNEEESSYSYEEINDQTNDN ITARLDRIDE KLSEILGMLH TLVVASAGPT SARDGIRDAM VGLREEMIEKIRTEALMTND RLEAMARLRN EESEKMAKDT SDEVSLNPTS EKLNNLLEGN DSDNDLSLED FNon-structural protein NS1 of the RSV strain ATCC VR-26 long:Amino acid sequence according to SEQ ID No. 10:MGSNSLSMIK VRLONLFDND EVALLKITCY TDKLIHLTNA LAKAVIHTIK LNGIVFVHVITSSDICPNNN IVVKSNFTTM PVLQNGGYIW EMMELTHCSQ PNGLIDDNCE IKFSKKLSDSTMTNYMNQLS ELLGFDLNPNon-structural protein NS2 of the RSV strain ATCC VR-26 long:Amino acid sequence according to SEQ ID No. 11:MDTTHNDTTP QRLMITDMRP LSLETTITSL TRDIITHRFI YLINHECIVR KLDERQATFTFLVNYEMKLL HKVGSTKYKK YTEYNTKYGT FPMPIFINHD GFLECIGIKP TKHTPIIYKY DLNP

In the context of the invention, additionally to the here disclosedamino acid sequences according to SEQ ID Nos. 1-11, also amino acidsequences of different Respiratory syncytial virus (RSV) isolates can beused according to the invention and are incorporated herewith. TheseRespiratory syncytial virus (RSV) isolates show preferably an identityof at least 70%, more preferably of at least 80% and most preferably ofat least 90% with the amino acid sequences according to SEQ ID Nos.1-11.

Furthermore, in this context the coding region encoding at least oneantigenic peptide or protein derived from the fusion protein F, theglycoprotein G, the short hydrophobic protein SH, the matrix protein M,the nucleoprotein N, the large polymerase L, the M2-1 protein, the M2-2protein, the phosphoprotein P, the non-structural protein NS1 or thenon-structural protein NS2 of Respiratory syncytial virus (RSV) or afragment, variant or derivative thereof, may be selected from anynucleic acid sequence comprising a coding region derived from anyRespiratory syncytial virus (RSV) isolate or a fragment or variantthereof.

Particularly preferred are the wild type mRNA sequences of the codingregions of the RSV strain long (ATCC VR-26) according to the NCBIaccession No. AY911262:

mRNA coding for the fusion protein F of the RSV strain ATCC VR-26 long:mRNA sequence according to SEQ ID No. 12:auggaguugccaauccucaaagcaaaugcaauuaccacaauccucgcugcagucacauuuugcuuugcuucuagucaaaacaucacugaagaauuuuaucaaucaacaugcagugcaguuagcaaaggcuaucuuagugcucuaagaacugguugguauacuaguguuauaacuauagaauuaaguaauaucaaggaaaauaaguguaauggaacagaugcuaagguaaaauugauaaaccaagaauuagauaaauauaaaaaugcuguaacagaauugcaguugcucaugcaaagcacaacagcagcaaacaaucgagccagaagagaacuaccaagguuuaugaauuauacacucaacaauaccaaaaaaaccaauguaacauuaagcaagaaaaggaaaagaagauuucuugguuuuuuguuagguguuggaucugcaaucgccaguggcauugcuguaucuaagguccugcacuuagaaggagaagugaacaagaucaaaagugcucuacuauccacaaacaaggccguagucagcuuaucaaauggaguuagugucuuaaccagcaaaguguuagaccucaaaaacuauauagauaaacaauuguuaccuauugugaauaagcaaagcugcagaauaucaaauauagaaacugugauagaguuccaacaaaagaacaacagacuacuagagauuaccagggaauuuaguguuaaugcagguguaacuacaccuguaagcacuuacauguuaacuaauagugaauuauugucauuaaucaaugauaugccuauaacaaaugaucagaaaaaguuaauguccaacaauguucaaauaguuagacagcaaaguuacucuaucauguccauaauaaaagaggaagucuuagcauauguaguacaauuaccacuauauggugugauagauacaccuuguuggaaauuacacacauccccucuauguacaaccaacacaaaagaagggucaaacaucuguuuaacaagaacugacagaggaugguacugugacaaugcaggaucaguaucuuucuucccacaagcugaaacauguaaaguucaaucgaaucgaguauuuugugacacaaugaacaguuuaacauuaccaagugaaguaaaucucugcaauguugacauauucaaucccaaauaugauuguaaaauuaugacuucaaaaacagauguaagcagcuccguuaucacaucucuaggagccauugugucaugcuauggcaaaacuaaauguacagcauccaauaaaaaucguggaaucauaaagacauuuucuaacgggugugauuauguaucaaauaaagggguggacacugugucuguagguaacacauuauauuauguaaauaagcaagaaggcaaaagucucuauguaaaaggugaaccaauaauaaauuucuaugacccauuaguauuccccucugaugaauuugaugcaucaauaucucaagucaaugagaagauuaaccagaguuuagcauuuauucguaaauccgaugaauuauuacaucauguaaaugcugguaaaucaaccacaaauaucaugauaacuacuauaauuauagugauuauaguaauauuguuaucauuaauugcuguuggacugcuccuauacuguaaggccagaagcacaccagucacacuaagcaaggaucaacugagugguauaaauaauauugcauuuaguaacugamRNA coding for the glycoprotein G of the RSV strain ATCC VR-26 long:mRNA sequence according to SEQ ID No. 13:auguccaaaaacaaggaccaacgcaccgcuaagacacuagaaaagaccugggacacucucaaucauuuuuauucauaucaucgggcuuauauaaguuaaaucuuaaaucuauagcacaaaucacauuauccauucuggcaaugauaaucucaacuucacuuauaauuacagccaucauauucauagccucggcaaaccacaaagucacacuaacaacugcaaucauacaagaugcaacaagccagaucaagaacacaaccccaacauaccucacucaggauccucagcuuggaaucagcuucuccaaucugucugaaauuacaucacaaaccaccaccauacuagcuucaacaacaccaggagucaagucaaaccugcaacccacaacagucaagacuaaaaacacaacaacaacccaaacacaacccagcaagcccacuacaaaacaacgccaaaacaaaccaccaaacaaacccaauaaugauuuucacuucgaaguguuuaacuuuguacccugcagcauaugcagcaacaauccaaccugcugggcuaucugcaaaagaauaccaaacaaaaaaccaggaaagaaaaccaccaccaagccuacaaaaaaaccaaccuucaagacaaccaaaaaagaucucaaaccucaaaccacuaaaccaaaggaaguacccaccaccaagcccacagaagagccaaccaucaacaccaccaaaacaaacaucacaacuacacugcucaccaacaacaccacaggaaauccaaaacucacaagucaaauggaaaccuuccacucaaccuccuccgaaggcaaucuaagcccuucucaagucuccacaacauccgagcacccaucacaacccucaucuccacccaacacaacacgccaguagmRNA coding for the Short hydrophobic protein SH of the RSV strain ATCC VR-26 long:mRNA sequence according to SEQ ID No. 14:auggaaaauacauccauaacaauagaauucucaagcaaauucuggccuuacuuuacacuaauacacaugaucacaacaauaaucucuuugcuaaucauaaucuccaucaugacugcaauacuaaacaaacuuugugaauauaacguauuccauaacaaaaccuuugaguuaccaagagcucgagucaacacauagmRNA coding for the matrixprotein M of the RSV strain ATCC VR-26 long:mRNA sequence according to SEQ ID No. 15:auggaaacauacgugaacaagcuucacgaaggcuccacauacacagcugcuguucaauacaauguccuagaaaaagacgaugacccugcaucacuuacaauaugggugcccauguuccaaucaucuaugccagcagauuuacuuauaaaagaacuagcuaaugucaacauacuagugaaacaaauauccacacccaagggaccuucacuaagagucaugauaaacucaagaagugcauugcuagcacaaaugcccagcaaauuuaccauaugugcuaauguguccuuggaugaaagaagcaaacuggcauaugauguaaccacacccugugaaaucaaggcauguagucuaacaugccuaaaaucaaaaaauauguuaacuacaguuaaagaucucacuaugaagacacucaaccccacacaugauauuauugcuuuaugugaauuugaaaacauaguaacaucaaaaaaagucauaauaccaacauaccuaagauccaucagugucagaaauaaagaucugaacacacuugaaaauauaacaaccacugaauucaaaaaugccaucacaaaugcaaaaaucaucccuuacucaggauuacuauuagucaucacagugacugacaacaaaggagcauucaaauacauaaagccgcaaagucaauucauaguagaucuuggagcuuaccuagaaaaagaaaguauauauuauguuaccacaaauuggaagcacacagcuacacgauuugcaaucaaacccauggaagauuaamRNA coding for the nucleoprotein N of the RSV strain ATCC VR-26 long:mRNA sequence according to SEQ ID No. 16:auggcucuuagcaaagucaaguugaaugauacacucaacaaagaucaacuucugucaucuagcaaauacaccauccaacggagcacaggagauaguauugauacuccuaauuaugaugugcagaaacacaucaauaaguuauguggcauguuauuaaucacagaagaugcuaaucauaaauucacuggguuaauagguauguuauaugcuaugucuagguuaggaagagaagacaccauaaaaauacucagagaugcgggauaucauguaaaagcaaauggaguagauguaacaacacaucgucaagacaucaaugggaaagaaaugaaauuugaaguguuaacauuggcaagcuuaacaacugaaauucaaaucaacauugagauagaaucuagaaaauccuacaaaaaaaugcuaaaagaaaugggagagguagcuccagaauacaggcaugauucuccugauugugggaugauaauauuauguauagcagcauuaguaauaaccaaauuggcagcaggggauagaucuggucuuacagccgugauuaggagagcuaauaauguccuaaaaaaugaaaugaaacguuacaaaggcuuacuacccaaggauauagccaacagcuucuaugaaguguuugaaaaacauccccacuuuauagauguuuuuguucauuuugguauagcacaaucuuccaccagagguggcaguagaguugaagggauuuuugcaggauuuuuaugaaugccuauggugcagggcaaguaaugcuacgguggggagucuuagcaaaaucaguuaaaaauauuauguuaggacaugcuagugugcaagcagaaauggaacaaguuguugagguuuaugaauaugcccaaaaauuggguggagaagcaggauucuaccauauauugaacaacccaaaagcaucauuauuaucuuugacucaauuuccucacuuuuccaguguaguauuaggcaaugcugcuggccuaggcauaaugggagaguacagagguacaccgaggaaucaagaucuauaugaugcagcaaaggcauaugcugaacaacucaaagaaaauggugugauuaacuacaguguauuagacuugacagcagaagaacuagaggcuaucaaacaucagcuuaauccaaaagauaaugauguagagcuuugamRNA coding for the Large polymerase L of the RSV strain ATCC VR-26 long:mRNA sequence according to SEQ ID No. 17:auggaucccauuauuaauggaaauucugcuaauguuuaucuaaccgauaguuauuuaaaagguguuaucucuuucucagaguguaaugcuuuaggaaguuacauauucaaugguccuuaucucaaaaaugauuauaccaacuuaauuaguagacaaaauccauuaauagaacacaugaaucuaaagaaacuaaauauaacacaguccuuaauaucuaaguaucauaaaggugaaaaaaauuagaagagccuacuuauuuucagucauuacuuaugacauacaagaguaugaccucguuggaacagauugcuaccacuaauuuacuuaaaaagauaauaagaagagcuauagaaauaagugaugucaaagucuaugcuauauugaauaaacuagggcuuaaagaaaaggacaagauuaaauccaacaauggacaggaugaagacaacucaguuauuacgaccauaaucaaagaugauauacuuucagcuguuaaggauaaucaaucucaucuuaaagcagacaaaaaucacucuacaaaacaaaaagacacaaucaaaacaacacucuugaagaaauuaauguguucaaugcagcauccuccaucaugguuaauacauugguuuaauuuauacacaaaauuaaacaacauauuaacacaguaucgaucaaaugagguuaaaaaccauggguuuauauugauagauaaucaaacucuuaguggauuucaauuuauuuugaaucaauaugguuguauaguuuaucauaaggaacucaaaagaauuacugugacaaccuauaaucaauucuugacauggaaagauauuagccuuaguagauuaaauguuuguuuaauuacauggauuaguaacugcuugaacacauuaaauaaaagcuuaggcuuaagaugcggauucaauaauguuaucuugacacaacuauuccuuuauggugauuguauacuaaagcuauuucacaaugagggguucuacauaauaaaagagguagagggauuuauuaugucucuaauuuuaaauauaacagaagaagaucaauucagaaaacgauuuuauaauaguaugcucaacaacaucacagaugcugcuaauaaagcucagaaaaaucugcuaucaagaguaugucauacauuauuagauaagacaguauccgauaauauaauaaauggcagauggauaauucuauuaaguaaguuccuuaaauuaauuaagcuugcaggugacaauaaccuuaacaaucugagugaacuauauuuuuuguucagaauauuuggacacccaaugguagaugaaagacaagccauggaugcuguuaaaguuaauugcaaugagaccaaauuuuacuuguuaagcaguuugaguauguuaagaggugccuuuauauauagaauuauaaaaggguuuguaaauaauuacaacagauggccuacuuuaagaaaugcuauuguuuuacccuuaagaugguuaacuuacuauaaacuaaacacuuauccuucuuuguuggaacuuacagaaagagauuugauuguguuaucaggacuacguuucuaucgugaguuucgguugccuaaaaaaguggaucuugaaaugauuauaaaugauaaagcuauaucacccccuaaaaauuugauauggacuaguuucccuagaaauuauaugccgucacacauacaaaacuauauagaacaugaaaaauuaaaauuuuccgagagugauaaaucaagaagaguauuagaguauuauuuaagagauaacaaauucaaugaaugugauuuauacaacuguguaguuaaucaaaguuaucucaacaacccuaaucaugugguaucauugacaggcaaagaaagagaacucaguguagguagaauguuugcaaugcaaccgggaauguucagacagguucaaauauuggcagagaaaaugauagcugaaaacauuuuacaauucuuuccugaaagucuuacaagauauggugaucuagaacuacaaaaaauauuagaauugaaagcaggaauaaguaacaaaucaaaucgcuacaaugauaauuacaacaauuacauuaguaagugcucuaucaucacagaucucagcaaauucaaucaagcauuucgauaugaaacgucauguauuuguagugaugugcuggaugaacugcaugguguacaaucucuauuuuccugguuacauuuaacuauuccucaugucacaauaauaugcacauauaggcaugcaccccccuauauaagagaucauauuguagaucuuaacaauguagaugaacaaaguggauuauauagauaucacaugggugguauugaaggguggugucaaaaacuauggaccauagaagcuauaucacuauuggaucuaauaucucucaaagggaaauucucaauuacugcuuuaauuaauggugacaaucaaucaauagauauaagcaaaccagucagacucauggaaggucaaacucaugcucaagcagauuauuugcuagcauuaaauagccuuaaauuacuguauaaagaguaugcaggcauaggucacaaauuaaaaggaacugagacuuauauaucacgagauaugcaauuuaugaguaaaacaauucaacauaacgguguauauuacccugcuaguauaaagaaaguccuaagagugggaccguggauaaacacuauacuugaugauuucaaagugagucuagaaucuauagguaguuugacacaagaauuagaauauagaggugaaagucuauuaugcaguuuaauauuuagaaauguaugguuauauaaucaaauugcucuacaauuaaaaaaucaugcguuauguaacaauaaauuauauuuggacauauuaaagguucugaaacacuuaaaaaccuuuuuuaaucuugauaauauugauacagcauuaacauuguauaugaauuuacccauguuauuuggugguggugaucccaacuuguuauaucgaaguuucuauagaagaacuccugauuuccucacagaggcuauaguucacucuguguucauacuuaguuauuauacaaaccaugacuuaaaagauaaacuucaagauuugucagaugauagauugaauaaguucuuaacaugcauaaucacguuugacaaaaacccuaaugcugaauucguaacauugaugagagauccucaagcuuuagggucugagagacaagcuaaaauuacuagugaaaucaauagacuggcaguuacagagguuuugaguacagcuccaaacaaaauauucuccaaaagugcacaacauuauaccacuacagagauagaucuaaaugauauuaugcaaaauauagaaccuacauauccucacgggcuaagaguuguuuaugaaaguuuacccuuuuauaaagcagagaaaauaguaaaucuuauaucagguacaaaaucuauaacuaacauacuggaaaagacuucugccauagacuuaacagauauugauagagccacugagaugaugaggaaaaacauaacuuugcuuauaaggauacuuccauuggauuguaacagagauaaaagagaaauauugaguauggaaaaccuaaguauuacugaauuaagcaaauauguuagggaaagaucuuggucuuuauccaauauaguugguguuacaucacccaguaucauguauacaauggacaucaaauauacaacaagcacuauagcuaguggcauaauuauagagaaauauaauguuaacaguuuaacacguggugagagaggaccaacuaaaccauggguugguucaucuacacaagagaaaaaaacaaugccaguuuauaauagacaaguuuuaaccaaaaaacaaagagaucaaauagaucuauuagcaaaauuggauuggguguaugcaucuauagauaacaaggaugaauucauggaagaacucagcauaggaacccuuggguuaacauaugaaaaggccaaaaaauuauuuccacaauauuuaagugucaacuauuugcaucgccuuacagucaguaguagaccaugugaauucccugcaucaauaccagcuuauagaacaacaaauuaucacuuugacacuagcccuauuaaucgcauauuaacagaaaaguauggugaugaagauauugacauaguauuccaaaacuguauaagcuuuggccuuagcuuaaugucaguaguagaacaauuuacuaauguauguccuaacagaauuauucucauaccuaagcuuaaugagauacauuugaugaaaccucccauauucacaggugauguugauauucacaaguuaaaacaagugauacaaaaacagcauauguuuuuaccagacaaaauaaguuugacucaauauguggaauuauucuuaaguaacaaaacacucaaaucuggaucucauguuaauucuaauuuaauauuggcacauaaaauaucugacuauuuucauaauacuuacauuuuaaguacuaauuuagcuggacauuggauucuaauuauacaacuuaugaaagauucuaaagguauuuuugaaaaagauuggggagagggauauauaacugaucauauguuuauuaauuugaaaguuuucuucaaugcuuauaagaccuaucucuuguguuuucauaaagguuauggcaaagcaaaacuggagugugauaugaacacuucagaucuucuauguguauuggaauuaauagacaguaguuauuggaagucuaugucuaagguauuuuuagaacaaaaaguuaucaaauacauucuuagccaagaugcaaguuuacauagaguaaaaggaugucauagcuucaaauuaugguuucuuaaacgucuuaauguagcagaauuuacaguuugcccuuggguuguuaacauagauuaucauccaacacauaugaaagcaauauuaacuuauauagaucuuguuagaaugggauugauaaauauagauagaauacacauuaaaaaaaacacaaauucaaugaugaauuuuauacuucuaaucucuuuuacauuaauuauaacuucucagauaauacucaucuauuaacuaaacauauaaggauugcuaauucagaauuagaaaauaauuacaacaaauuauaucauccuacaccagaaacccuagagaauauacuagccaauccgauuaaaaguaaugacaaaaagacacugaacgacuauuguauagguaaaaauguugacucaauaauguuaccauuguuaucuaauaagaagcuuguuaaaucgucugcaaugauuagaaccaauuacagcaaacaagaccuguacaaucuauucccuacgguugugaucgauagaauuauagaucauucagguaauacagccaaauccaaccaacuuuacacuacuacuucccaucaaauaucuuuagugcacaauagcacaucacuuuauugcaugcuuccuuggcaucauauuaauagauucaauuuuguauuuaguucuacagguuguaaaauuaguauagaguauauuuuaaaagaccuuaaaauuaaagauccuaauuguauagcauucauaggugaaggagcagggaauuuauuauugcguacagugguggaacuucauccugacauaagauauauuuacagaagucugaaagauugcaaugaucauaguuuaccuauugaguuuuuaaggcuauacaauggacauaucaacauugauuauggugaaaauuugaccauuccugcuacagaugcaaccaacaacauucauuggucuuauuuacauauaaaguuugcugaaccuaucagucuuuuuguaugugaugccgaauugccuguaacagucaacuggaguaaaauuauaauagaauggagcaagcauguaagaaaaugcaaguacuguuccucaguuaauaaauguacguuaauaguaaaauaucaugcucaagaugauauugauuucaaauuagacaauauaacuauauuaaaaacuuauguaugcuuaggcaguaaguuaaagggaucggagguuuacuuaguccuuacaauagguccugcaaauauauuuccaguauuuaauguaguacaaaaugcuaaauugauacuaucaagaaccaaaaauuucaucaugccuaagaaagcugauaaagagucuauugaugcaaauauuaaaaguuugauacccuuuuuuguuaccuauaacaaaaaaaggaauuaauacugcauugucaaaacuaaagaguguuguuaguggagauauacuaucauauucuauagcuggacggaaugaaguuuucagcaauaaacuuauaaaucauaagcauaugaacaucuuaaagugguucaaucauguuuuaaauuucagaucaacagaacuaaacuauaaccauuuauauaugguagaaucuacauauccuuaccuaagugaauuguuaaacagcuugacaacuaaugaacuuaaaaaacugauuaaaaucacagguagucuguuauacaacuuucauaaugaauaamRNA coding for the protein M2-1 of the RSV strain ATCC VR-26 long:mRNA sequence according to SEQ ID No. 18:AugucacgaaggaauccuugcaaauuugaaauucgaggucauugcuugaaugguaagagaugucauuuuagucauaauuauuuugaauggccaccccaugcacugcucguaagacaaaacuuuauguuaaacagaauacuuaagucuauggauaaaaguauagauaccuuaucagaaauaaguggagcugcagaguuggacagaacagaagaguaugcucuugguguaguuggagugcuagagaguuauauaggaucaauaaauaauauaacuaaacaaucagcauguguugccaugagcaaacuccucacugaacucaauagugaugauaucaaaaaacugagagacaaugaagagcuaaauucacccaagauaagaguguacaauacugucauaucauauauugaaagcaacaggaaaaacaauaaacaaacuauccaucuguuaaaaagauugccagcagacguauugaagaaaaccaucaaaaacacauuggauauccacaagagcauaaccaucaacaacccaaaagaauuaacuguuagugauacaaaugaccaugccaaaaauaaugauacuaccugamRNA coding for the protein M2-2 of the RSV strain ATCC VR-26 long:mRNA sequence according to SEQ ID No. 19:augaccaugccaaaaauaaugauacuaccugacaaauauccuuguaguauaacuuccauacuaauaacaaguagauguagagucacuauguauaaucgaaagaacacacuauauuucaaucaaaacaacccaaauaaccauauguacucaccgaaucaaacauucaaugaaauccauuggaccucacaagacuugauugacacaauucaaaauuuucuacagcaucuagguguuauugaggauauauauacaauauauauauuagugucauaamRNA coding for the phosphoprotein P of the RSV strain ATCC VR-26 long:mRNA sequence according to SEQ ID No. 20:auggaaaaguuugcuccugaauuccauggagaagaugcaaacaacagggcuacuaaauuccuagaaucaauaaagggcaaauucacaucaccuaaagaucccaagaaaaaagauaguaucauaucugucaacucaauagauauagaaguaaccaaagaaagcccuauaacaucaaauucaaccauuauuaacccaacaaaugagacagaugauaaugcagggaacaagcccaauuaucaaagaaaaccucuaguaaguuucaaagaagacccuauaccaagugauaaucccuuuucaaaacuauacaaagaaaccauagagacauuugauaacaaugaagaagaaucuagcuauucauaugaagaaauaaaugaucagacgaacgauaauauaacugcaagauuagauaggauugaugaaaaauuaagugaaauacuaggaaugcuucacacauuaguaguagcaagugcaggaccuacaucugcuagggaugguauaagagaugccaugguugguuuaagagaagaaaugauagaaaaaaucagaacugaagcauuaaugaccaaugacagauuagaagcuauggcaagacucaggaaugaggaaagugaaaagauggcaaaagacacaucagaugaagugucucucaauccaacaucagagaaauugaacaaccuguuggaagggaaugauagugacaaugaucuaucacuugaagauuucugamRNA coding for the non-structural protein NS1 of the RSV strain ATCC VR-26 long:mRNA sequence according to SEQ ID No. 21:augggcagcaauucguugaguaugauaaaaguuagauuacaaaauuuguuugacaaugaugaaguagcauuguuaaaaauaacaugcuauacugacaaauuaauacauuuaacuaaugcuuuggcuaaggcagugauacauacaaucaaauugaauggcauuguguuugugcauguuauuacaaguagugauauuugcccuaauaauaauauuguaguaaaauccaauuucacaacaaugccagugcuacaaaauggagguuauauaugggaaaugauggaauuaacacauugcucucaaccuaauggucuaauagaugacaauugugaaauuaaauucuccaaaaaacuaagugauucaacaaugaccaauuauaugaaucaauuaucugaauuacuuggauuugaucuuaauccauaamRNA coding for the non-structural protein NS2 of the RSV strain ATCC VR-26 long:mRNA sequence according to SEQ ID No. 22:auggacacaacccacaaugauaccacaccacaaagacugaugaucacagacaugagaccguugucacuugagacuacaauaacaucacuaaccagagacaucauaacacacagauuuauauacuuaauaaaucaugaaugcauagugagaaaacuugaugaaagacaggccacauuuacauuccuggucaacuaugaaaugaaacuauugcacaaaguaggaagcacuaaauauaaaaaauauacugaauacaacacaaaauauggcacuuucccuaugccgauauucaucaaucaugauggguucuuagaaugcauuggcauuaagccuacaaagcauacucccauaauauacaaguaugaucucaauccauag

In the context of the invention, additionally to the here disclosednucleic acid sequences, also nucleic acid sequences of differentRespiratory syncytial virus (RSV) isolates are incorporated herewith.These different Respiratory syncytial virus (RSV) isolates showpreferably an identity of at least 50%, 60%, 70%, more preferably of atleast 80% and most preferably of at least 90% with the nucleic acidsequences according to SEQ ID Nos. 12-22 or of fragments thereof.

In a preferred embodiment, the mRNA sequence according to the inventiondoes not comprise a reporter gene or a marker gene. Preferably, the mRNAsequence according to the invention does not encode, for instance,luciferase; green fluorescent protein (GFP) and its variants (such aseGFP, RFP or BFP); α-globin; hypoxanthine-guaninephosphoribosyltransferase (HGPRT); β-galactosidase; galactokinase;alkaline phosphatase; secreted embryonic alkaline phosphatase (SEAP)) ora resistance gene (such as a resistance gene against neomycin,puromycin, hygromycin and zeocin). In a preferred embodiment, the mRNAsequence according to the invention does not encode luciferase. Inanother embodiment, the mRNA sequence according to the invention doesnot encode GFP or a variant thereof.

In a further preferred embodiment, the mRNA sequence according to theinvention does not encode a protein (or a fragment of a protein) derivedfrom a virus belonging to the family of Orthomyxoviridae. Preferably themRNA sequence does not encode a protein that is derived from aninfluenza virus, more preferably an influenza A virus. Preferably, themRNA sequence according to the invention does not encode an influenza Aprotein selected from the group consisting of hemagglutinin (HA),neuraminidase (NA), nucleoprotein (NP), M1, M2, NS1, NS2 (NEP: nuclearexport protein), PA, PB1 (polymerase basic 1), PB1-F2 and PB2. Inanother preferred embodiment, the mRNA according to the invention doesnot encode ovalbumin (OVA) or a fragment thereof. Preferably, the mRNAsequence according to the invention does not encode an influenza Aprotein or ovalbumin.

By a further embodiment, the inventive mRNA preferably comprises atleast one of the following structural elements: a 5′- and/or3′-untranslated region element (UTR element), particularly a 5′-UTRelement which comprises or consists of a nucleic acid sequence which isderived from the 5′-UTR of a TOP gene or from a fragment, homolog or avariant thereof, or a 5′- and/or 3′-UTR element which may be derivablefrom a gene that provides a stable mRNA or from a homolog, fragment orvariant thereof; a histone-stem-loop structure, preferably ahistone-stem-loop in its 3′ untranslated region; a 5′-CAP structure; apoly-A tail; or a poly(C) sequence.

In a preferred embodiment of the first aspect of the present invention,the inventive mRNA comprises at least one 5′- or 3′-UTR element. In thiscontext, an UTR element comprises or consists of a nucleic acid sequencewhich is derived from the 5′- or 3′-UTR of any naturally occurring geneor which is derived from a fragment, a homolog or a variant of the 5′-or 3′-UTR of a gene. Preferably, the 5′- or 3′-UTR element usedaccording to the present invention is heterologous to the coding regionof the inventive mRNA sequence. Even if 5′- or 3′-UTR elements derivedfrom naturally occurring genes are preferred, also syntheticallyengineered UTR elements may be used in the context of the presentinvention.

In a particularly preferred embodiment of the first aspect of thepresent invention, the inventive mRNA sequence comprises at least one5′-untranslated region element (5′UTR element) which comprises orconsists of a nucleic acid sequence which is derived from the 5′UTR of aTOP gene or which is derived from a fragment, homolog or variant of the5′UTR of a TOP gene.

It is particularly preferred that the 5′UTR element does not comprise aTOP-motif or a 5′TOP, as defined above.

In some embodiments, the nucleic acid sequence of the 5′UTR elementwhich is derived from a 5′UTR of a TOP gene terminates at its 3′-endwith a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10upstream of the start codon (e.g. A(U/T)G) of the gene or mRNA it isderived from. Thus, the 5′UTR element does not comprise any part of theprotein coding region. Thus, preferably, the only protein coding part ofthe inventive mRNA is provided by the coding region.

The nucleic acid sequence, which is derived from the 5′UTR of a TOPgene, is derived from a eukaryotic TOP gene, preferably a plant oranimal TOP gene, more preferably a chordate TOP gene, even morepreferably a vertebrate TOP gene, most preferably a mammalian TOP gene,such as a human TOP gene.

For example, the 5′UTR element is preferably selected from 5′-UTRelements comprising or consisting of a nucleic acid sequence, which isderived from a nucleic acid sequence selected from the group consistingof SEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO.1422 of the patent application WO2013/143700, whose disclosure isincorporated herein by reference, from the homologs of SEQ ID Nos.1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of thepatent application WO2013/143700, from a variant thereof, or preferablyfrom a corresponding RNA sequence. The term “homologs of SEQ ID Nos.1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of thepatent application WO2013/143700” refers to sequences of other speciesthan Homo sapiens, which are homologous to the sequences according toSEQ ID Nos. 1-1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422of the patent application WO2013/143700.

In a preferred embodiment, the 5′UTR element comprises or consists of anucleic acid sequence which is derived from a nucleic acid sequenceextending from nucleotide position 5 (i.e. the nucleotide that islocated at position 5 in the sequence) to the nucleotide positionimmediately 5′ to the start codon (located at the 3′ end of thesequences), e.g. the nucleotide position immediately 5′ to the ATGsequence, of a nucleic acid sequence selected from SEQ ID Nos. 1-1363,SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700, from the homologs of SEQ ID Nos. 1-1363, SEQID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700, from a variant thereof, or a correspondingRNA sequence. It is particularly preferred that the 5′ UTR element isderived from a nucleic acid sequence extending from the nucleotideposition immediately 3′ to the 5′TOP to the nucleotide positionimmediately 5′ to the start codon (located at the 3′ end of thesequences), e.g. the nucleotide position immediately 5′ to the ATGsequence, of a nucleic acid sequence selected from SEQ ID Nos. 1-1363,SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700, from the homologs of SEQ ID Nos. 1-1363, SEQID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patentapplication WO2013/143700, from a variant thereof, or a correspondingRNA sequence.

In a particularly preferred embodiment, the 5′UTR element comprises orconsists of a nucleic acid sequence, which is derived from a 5′UTR of aTOP gene encoding a ribosomal protein or from a variant of a 5′UTR of aTOP gene encoding a ribosomal protein. For example, the 5′UTR elementcomprises or consists of a nucleic acid sequence which is derived from a5′UTR of a nucleic acid sequence according to any of SEQ ID NOs: 67,170, 193, 244, 259, 554, 650, 675, 700, 721, 913, 1016, 1063, 1120,1138, and 1284-1360 of the patent application WO2013/143700, acorresponding RNA sequence, a homolog thereof, or a variant thereof asdescribed herein, preferably lacking the 5′TOP motif. As describedabove, the sequence extending from position 5 to the nucleotideimmediately 5′ to the ATG (which is located at the 3′end of thesequences) corresponds to the 5′UTR of said sequences.

Preferably, the 5′UTR element comprises or consists of a nucleic acidsequence which is derived from a 5′UTR of a TOP gene encoding aribosomal Large protein (RPL) or from a homolog or variant of a 5′UTR ofa TOP gene encoding a ribosomal Large protein (RPL). For example, the5′UTR element comprises or consists of a nucleic acid sequence which isderived from a 5′UTR of a nucleic acid sequence according to any of SEQID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1358, 1421 and1422 of the patent application WO2013/143700, a corresponding RNAsequence, a homolog thereof, or a variant thereof as described herein,preferably lacking the 5′ TOP motif.

In a particularly preferred embodiment, the 5′UTR element comprises orconsists of a nucleic acid sequence which is derived from the 5′UTR of aribosomal protein Large 32 gene, preferably from a vertebrate ribosomalprotein Large 32 (L32) gene, more preferably from a mammalian ribosomalprotein Large 32 (L32) gene, most preferably from a human ribosomalprotein Large 32 (L32) gene, or from a variant of the 5′UTR of aribosomal protein Large 32 gene, preferably from a vertebrate ribosomalprotein Large 32 (L32) gene, more preferably from a mammalian ribosomalprotein Large 32 (L32) gene, most preferably from a human ribosomalprotein Large 32 (L32) gene, wherein preferably the 5′UTR element doesnot comprise the 5′TOP of said gene.

Accordingly, in a particularly preferred embodiment, the 5′UTR elementcomprises or consists of a nucleic acid sequence which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID No. 23 (5′-UTR ofhuman ribosomal protein Large 32 lacking the 5′ terminal oligopyrimidinetract: GGCGCTGCCTACGGAGGTGGCAGCCATCTCCTTCTCGGCATC; corresponding to SEQID No. 1368 of the patent application WO2013/143700) or preferably to acorresponding RNA sequence, or wherein the at least one 5′UTR elementcomprises or consists of a fragment of a nucleic acid sequence which hasan identity of at least about 40%, preferably of at least about 50%,preferably of at least about 60%, preferably of at least about 70%, morepreferably of at least about 80%, more preferably of at least about 90%,even more preferably of at least about 95%, even more preferably of atleast about 99% to the nucleic acid sequence according to SEQ ID No. 23or more preferably to a corresponding RNA sequence, wherein, preferably,the fragment is as described above, i.e. being a continuous stretch ofnucleotides representing at least 20%, preferably at least 30%, morepreferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, even more preferably at least 70%, even morepreferably at least 80%, and most preferably at least 90%. of thefull-length 5′UTR. Preferably, the fragment exhibits a length of atleast about 20 nucleotides or more, preferably of at least about 30nucleotides or more, more preferably of at least about 40 nucleotides ormore. Preferably, the fragment is a functional fragment as describedherein.

In some embodiments, the inventive mRNA comprises a 5′UTR element whichcomprises or consists of a nucleic acid sequence which is derived fromthe 5′UTR of a vertebrate TOP gene, such as a mammalian, e.g. a humanTOP gene, selected from RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7,RPS8, RPS9, RPS10, RPS11, RPS12, RPS13, RPS14, RPS15, RPS15A, RPS16,RPS17, RPS18, RPS19, RPS20, RPS21, RPS23, RPS24, RPS25, RPS26, RPS27,RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8,RPL9, RPL10, RPL10A, RPL11, RPL12, RPL13, RPL13A, RPL14, RPL15, RPL17,RPL18, RPL18A, RPL19, RPL21, RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27,RPL27A, RPL28, RPL29, RPL30, RPL31, RPL32, RPL34, RPL35, RPL35A, RPL36,RPL36A, RPL37, RPL37A, RPL38, RPL39, RPL40, RPL41, RPLP0, RPLP1, RPLP2,RPLP3, RPLP0, RPLP1, RPLP2, EEF1A1, EEF1B2, EEF1D, EEF1G, EEF2, EIF3E,EIF3F, EIF3H, EIF2S3, EIF3C, EIF3K, EIF3EIP, EIF4A2, PABPC1, HNRNPA1,TPT1, TUBB 1, UBA52, NPM1, ATP5G2, GNB2L1, NME2, UQCRB, or from ahomolog or variant thereof, wherein preferably the 5′UTR element doesnot comprise a TOP-motif or the 5′TOP of said genes, and whereinoptionally the 5′UTR element starts at its 5′-end with a nucleotidelocated at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 downstream of the5′terminal oligopyrimidine tract (TOP) and wherein further optionallythe 5′UTR element which is derived from a 5′UTR of a TOP gene terminatesat its 3′-end with a nucleotide located at position 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 upstream of the start codon (A(U/T)G) of the gene it isderived from.

In further particularly preferred embodiments, the 5′UTR elementcomprises or consists of a nucleic acid sequence which is derived fromthe 5′UTR of a ribosomal protein Large 32 gene (RPL32), a ribosomalprotein Large 35 gene (RPL35), a ribosomal protein Large 21 gene(RPL21), an ATP synthase, H+ transporting, mitochondrial F1 complex,alpha subunit 1, cardiac muscle (ATP5A1) gene, an hydroxysteroid(17-beta) dehydrogenase 4 gene (HSD17B4), an androgen-induced 1 gene(AIG1), cytochrome c oxidase subunit VIc gene (COX6C), or aN-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1) orfrom a variant thereof, preferably from a vertebrate ribosomal proteinLarge 32 gene (RPL32), a vertebrate ribosomal protein Large 35 gene(RPL35), a vertebrate ribosomal protein Large 21 gene (RPL21), avertebrate ATP synthase, H+ transporting, mitochondrial F1 complex,alpha subunit 1, cardiac muscle (ATP5A1) gene, a vertebratehydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4), a vertebrateandrogen-induced 1 gene (AIG1), a vertebrate cytochrome c oxidasesubunit VIc gene (COX6C), or a vertebrate N-acylsphingosineamidohydrolase (acid ceramidase) 1 gene (ASAH1) or from a variantthereof, more preferably from a mammalian ribosomal protein Large 32gene (RPL32), a ribosomal protein Large 35 gene (RPL35), a ribosomalprotein Large 21 gene (RPL21), a mammalian ATP synthase, H+transporting, mitochondrial F1 complex, alpha subunit 1, cardiac muscle(ATP5A1) gene, a mammalian hydroxysteroid (17-beta) dehydrogenase 4 gene(HSD17B4), a mammalian androgen-induced 1 gene (AIG1), a mammaliancyto-chrome c oxidase subunit VIc gene (COX6C), or a mammalianN-acylsphingosine ami-dohydrolase (acid ceramidase) 1 gene (ASAH1) orfrom a variant thereof, most preferably from a human ribosomal proteinLarge 32 gene (RPL32), a human ribosomal protein Large 35 gene (RPL35),a human ribosomal protein Large 21 gene (RPL21), a human ATP syn-thase,H+ transporting, mitochondrial F1 complex, alpha subunit 1, cardiacmuscle (ATP5A1) gene, a human hydroxysteroid (17-beta) dehydrogenase 4gene (HSD17B4), a human androgen-induced 1 gene (AIG1), a humancytochrome c oxidase subunit VIc gene (COX6C), or a humanN-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1) orfrom a variant thereof, wherein preferably the 5′UTR element does notcomprise the 5′TOP of said gene.

Accordingly, in a particularly preferred embodiment, the 5′UTR elementcomprises or consists of a nucleic acid sequence which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID No. 1368, or SEQ IDNOs 1412-1420 of the patent application WO2013/143700, or acorresponding RNA sequence, or wherein the at least one 5′UTR elementcomprises or consists of a fragment of a nucleic acid sequence which hasan identity of at least about 40%, preferably of at least about 50%,preferably of at least about 60%, preferably of at least about 70%, morepreferably of at least about 80%, more preferably of at least about 90%,even more preferably of at least about 95%, even more preferably of atleast about 99% to the nucleic acid sequence according to SEQ ID No.1368, or SEQ ID NOs 1412-1420 of the patent application WO2013/143700,wherein, preferably, the fragment is as described above, i.e. being acontinuous stretch of nucleotides representing at least 20% etc. of thefull-length 5′UTR. Preferably, the fragment exhibits a length of atleast about 20 nucleotides or more, preferably of at least about 30nucleotides or more, more preferably of at least about 40 nucleotides ormore. Preferably, the fragment is a functional fragment as describedherein.

Accordingly, in a particularly preferred embodiment, the 5′UTR elementcomprises or consists of a nucleic acid sequence which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID No. 36 (5′-UTR ofATP5A1 lacking the 5′ terminal oligopyrimidine tract:GCGGCTCGGCCATTTTGTCCCAGTCAGTCCGGAGGCTGCGGCTGCAGAAGTACCGCCTGCG-GAGTAACTGCAAAG; corresponding to SEQ ID No. 1414 of the patentapplication WO2013/143700) or preferably to a corresponding RNAsequence, or wherein the at least one 5′UTR element comprises orconsists of a fragment of a nucleic acid sequence which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID No. 26 or morepreferably to a corresponding RNA sequence, wherein, preferably, thefragment is as described above, i.e. being a continuous stretch ofnucleotides representing at least 20% etc. of the full-length 5′UTR.Preferably, the fragment exhibits a length of at least about 20nucleotides or more, preferably of at least about 30 nucleotides ormore, more preferably of at least about 40 nucleotides or more.Preferably, the fragment is a functional fragment as described herein.

In a further preferred embodiment, the inventive mRNA further comprisesat least one 3′UTR element, which comprises or consists of a nucleicacid sequence derived from the 3′UTR of a chordate gene, preferably avertebrate gene, more preferably a mammalian gene, most preferably ahuman gene, or from a variant of the 3′UTR of a chordate gene,preferably a vertebrate gene, more preferably a mammalian gene, mostpreferably a human gene.

The term ‘3′UTR element’ refers to a nucleic acid sequence whichcomprises or consists of a nucleic acid sequence that is derived from a3′UTR or from a variant of a 3′UTR. A 3′UTR element in the sense of thepresent invention may represent the 3′UTR of an mRNA. Thus, in the senseof the present invention, preferably, a 3′UTR element may be the 3′UTRof an mRNA, preferably of an artificial mRNA, or it may be thetranscription template for a 3′UTR of an mRNA. Thus, a 3′UTR elementpreferably is a nucleic acid sequence which corresponds to the 3′UTR ofan mRNA, preferably to the 3′UTR of an artificial mRNA, such as an mRNAobtained by transcription of a genetically engineered vector construct.Preferably, the 3′UTR element fulfils the function of a 3′UTR or encodesa sequence which fulfils the function of a 3′UTR.

Preferably, the inventive mRNA comprises a 3′UTR element which may bederivable from a gene that relates to an mRNA with an enhanced half-life(that provides a stable mRNA), for example a 3′UTR element as definedand described below.

In a particularly preferred embodiment, the 3′UTR element comprises orconsists of a nucleic acid sequence which is derived from a 3′UTR of agene selected from the group consisting of an albumin gene, an α-globingene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene,and a collagen alpha gene, such as a collagen alpha 1(I) gene, or from avariant of a 3′UTR of a gene selected from the group consisting of analbumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylasegene, a lipoxygenase gene, and a collagen alpha gene, such as a collagenalpha 1(I) gene according to SEQ ID No. 1369-1390 of the patentapplication WO2013/143700 whose disclosure is incorporated herein byreference. In a particularly preferred embodiment, the 3′UTR elementcomprises or consists of a nucleic acid sequence which is derived from a3′UTR of an albumin gene, preferably a vertebrate albumin gene, morepreferably a mammalian albumin gene, most preferably a human albumingene according to SEQ ID No. 24.

Human albumin 3′UTR SEQ ID No. 24:CATCACATTT AAAAGCATCT CAGCCTACCA TGAGAATAAGAGAAAGAAAA TGAAGATCAA AAGCTTATTC ATCTGTTTTTCTTTTTCGTT GGTGTAAAGC CAACACCCTG TCTAAAAAACATAAATTTCT TTAATCATTT TGCCTCTTTT CTCTGTGCTTCAATTAATAA AAAATGGAAA GAATCT (correspondingto SEQ ID No: 1369 of the patent application WO2013/143700).

In this context, it is particularly preferred that the inventive mRNAcomprises a 3′-UTR element comprising a corresponding RNA sequencederived from the nucleic acids according to SEQ ID No. 1369-1390 of thepatent application WO2013/143700 or a fragment, homolog or variantthereof.

Most preferably the 3′-UTR element comprises the nucleic acid sequencederived from a fragment of the human albumin gene according to SEQ IDNo. 25:

albumin? 3′UTR

CATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCT (SEQ IDNo. 25 corresponding to SEQ ID No: 1376 of thepatent application WO2013/143700)

In this context, it is particularly preferred that the 3′-UTR element ofthe inventive mRNA comprises or consists of a corresponding RNA sequenceof the nucleic acid sequence according to SEQ ID No. 25.

In another particularly preferred embodiment, the 3′UTR elementcomprises or consists of a nucleic acid sequence which is derived from a3′UTR of an α-globin gene, preferably a vertebrate α- or β-globin gene,more preferably a mammalian α- or β-globin gene, most preferably a humanα- or β-globin gene according to SEQ ID No. 26-28:

3′-UTR of Homo sapiens hemoglobin, alpha 1 (HBA1)GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ IDNo:26 corresponding to SEQ ID No. 1370 of the patent application WO2013/143700)3′-UTR of Homo sapiens hemoglobin, alpha 2 (HBA2)GCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCTGGTCTTTGAATAAAGTCTGAGTGGGCAG (SEQ IDNo: 27 corresponding to SEQ ID No. 1371 of the patent application WO2013/143700)3′-UTR of Homo sapiens hemoglobin, beta (HBB)GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC (SEQ ID No: 28 corresponding to SEQ ID No. 1372 of the patentapplication WO2013/143700)

For example, the 3′UTR element may comprise or consist of the center,α-complex-binding portion of the 3′UTR of an α-globin gene, such as of ahuman α-globin gene, preferably according to SEQ ID No. 29:

Center, α-complex-binding portion of the 3′UTR of an α-globin gene (alsonamed herein as “muag”)

GCCCGATGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCG (SEQ ID NO. 29 corresponding to SEQ ID No. 1393 of the patent application WO2013/143700).

In this context, it is particularly preferred that the 3′-UTR element ofthe inventive mRNA comprises or consists of a corresponding RNA sequenceof the nucleic acid sequence according to SEQ ID No. 29 or a homolog, afragment or variant thereof.

The term ‘a nucleic acid sequence which is derived from the 3′UTR of a [. . . ] gene’ preferably refers to a nucleic acid sequence which isbased on the 3′UTR sequence of a [ . . . ] gene or on a part thereof,such as on the 3′UTR of an albumin gene, an α-globin gene, a β-globingene, a tyrosine hydroxylase gene, a lipoxygenase gene, or a collagenalpha gene, such as a collagen alpha 1(I) gene, preferably of an albumingene or on a part thereof. This term includes sequences corresponding tothe entire 3′UTR sequence, i.e. the full length 3′UTR sequence of agene, and sequences corresponding to a fragment of the 3′UTR sequence ofa gene, such as an albumin gene, α-globin gene, β-globin gene, tyrosinehydroxylase gene, lipoxygenase gene, or collagen alpha gene, such as acollagen alpha 1(I) gene, preferably of an albumin gene.

The term ‘a nucleic acid sequence which is derived from a variant of the3′UTR of a [ . . . ] gene’ preferably refers to a nucleic acid sequencewhich is based on a variant of the 3′UTR sequence of a gene, such as ona variant of the 3′UTR of an albumin gene, an α-globin gene, a β-globingene, a tyrosine hydroxylase gene, a lipoxygenase gene, or a collagenalpha gene, such as a collagen alpha 1(I) gene, or on a part thereof asdescribed above. This term includes sequences corresponding to theentire sequence of the variant of the 3′UTR of a gene, i.e. the fulllength variant 3′UTR sequence of a gene, and sequences corresponding toa fragment of the variant 3′UTR sequence of a gene. A fragment in thiscontext preferably consists of a continuous stretch of nucleotidescorresponding to a continuous stretch of nucleotides in the full-lengthvariant 3′UTR, which represents at least 20%, preferably at least 30%,more preferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, even more preferably at least 70%, even morepreferably at least 80%, and most preferably at least 90% of thefull-length variant 3′UTR. Such a fragment of a variant, in the sense ofthe present invention, is preferably a functional fragment of a variantas described herein.

Preferably, the at least one 5′UTR element and the at least one 3′UTRelement act synergistically to increase protein production from theinventive mRNA as described above.

In a particularly preferred embodiment, the inventive mRNA comprising acoding region, encoding at least one antigenic peptide or protein ofRespiratory syncytial virus (RSV) or a fragment, variant or derivativethereof, comprises a histone stem-loop sequence/structure. Such histonestem-loop sequences are preferably selected from histone stem-loopsequences as disclosed in WO 2012/019780, whose disclosure isincorporated herewith by reference.

A histone stem-loop sequence, suitable to be used within the presentinvention, is preferably selected from at least one of the followingformulae (I) or (II):

Formula (I) (Stem-Loop Sequence without Stem Bordering Elements):

Formula (II) (Stem-Loop Sequence with Stem Bordering Elements):

wherein:

stem1 or stem2 bordering elements is a consecutive sequence of 1 to 6,preferably of 2 to N₁₋₆ 6, more preferably of 2 to 5, even morepreferably of 3 to 5, most preferably of 4 to 5 or 5 N, wherein each Nis independently from another selected from a nucleotide selected fromA, U, T, G and C, or a nucleotide analogue thereof; stem1 [N₀₋₂GN₃₋₅] isreverse complementary or partially reverse complementary with elementstem2, and is a consecutive sequence between of 5 to 7 nucleotides;wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of 0 to 1,more preferably of 1 N, wherein each N is independently from anotherselected from a nucleotide selected from A, U, T, G and C or anucleotide analogue thereof; wherein N₃₋₅ is a consecutive sequence of 3to 5, preferably of 4 to 5, more preferably of 4 N, wherein each N isindependently from another selected from a nucleotide selected from A,U, T, G and C or a nucleotide analogue thereof, and wherein G isguanosine or an analogue thereof, and may be optionally replaced by acytidine or an analogue thereof, provided that its complementarynucleotide cytidine in stem2 is replaced by guanosine; loop sequence[N₀₋₄(U/T)N₀₋₄] is located between elements stem1 and stem2, and is aconsecutive sequence of 3 to 5 nucleotides, more preferably of 4nucleotides; wherein each N₀₋₄ is independent from another a consecutivesequence of 0 to 4, preferably of 1 to 3, more preferably of 1 to 2 N,wherein each N is independently from another selected from a nucleotideselected from A, U, T, G and C or a nucleotide analogue thereof; andwherein U/T represents uridine, or optionally thymidine; stem2[N₃₋₅CN₀₋₂] is reverse complementary or partially reverse complementarywith element stem1, and is a consecutive sequence between of 5 to 7nucleotides; wherein N₃₋₅ is a consecutive sequence of 3 to 5,preferably of 4 to 5, more preferably of 4 N, wherein each N isindependently from another selected from a nucleotide selected from A,U, T, G and C or a nucleotide analogue thereof; wherein N₀₋₂ is aconsecutive sequence of 0 to 2, preferably of 0 to 1, more preferably of1 N, wherein each N is independently from another selected from anucleotide selected from A, U, T, G or C or a nucleotide analoguethereof; and wherein C is cytidine or an analogue thereof, and may beoptionally replaced by a guanosine or an analogue thereof provided thatits complementary nucleoside guanosine in stem1 is replaced by cytidine;

wherein

stem1 and stem2 are capable of base pairing with each other forming areverse complementary sequence, wherein base pairing may occur betweenstem1 and stem2, e.g. by Watson-Crick base pairing of nucleotides A andU/T or G and C or by non-Watson-Crick base pairing e.g. wobble basepairing, reverse Watson-Crick base pairing, Hoogsteen base pairing,reverse Hoogsteen base pairing or are capable of base pairing with eachother forming a partially reverse complementary sequence, wherein anincomplete base pairing may occur between stem1 and stem2, on the basisthat one ore more bases in one stem do not have a complementary base inthe reverse complementary sequence of the other stem.

According to a further preferred embodiment of the first inventiveaspect, the inventive mRNA sequence may comprise at least one histonestem-loop sequence according to at least one of the following specificformulae (Ia) or (IIa):

Formula (Ia) (Stem-Loop Sequence without Stem Bordering Elements):

Formula (IIa) (Stem-Loop Sequence with Stem Bordering Elements):

wherein:

-   -   N, C, G, T and U are as defined above.

According to a further more particularly preferred embodiment of thefirst aspect, the inventive mRNA sequence may comprise at least onehistone stem-loop sequence according to at least one of the followingspecific formulae (Ib) or (IIb):

Formula (Ib) (Stem-Loop Sequence without Stem Bordering Elements):

Formula (IIb) (Stem-Loop Sequence with Stem Bordering Elements):

wherein:

-   -   N, C, G, T and U are as defined above.

A particular preferred histone stem-loop sequence is the sequenceaccording to SEQ ID NO: 30 CAAAGGCTCTTTTCAGAGCCACCA or more preferablythe corresponding RNA sequence of the nucleic acid sequence according toSEQ ID NO: 30 (CAAAGGCUCUUUUCAGAGCCACCA SEQ ID NO: 37).

In a particular preferred embodiment of the first aspect of the presentinvention the inventive mRNA comprises additionally to the coding regionencoding at least one antigenic peptide or protein of Respiratorysyncytial virus (RSV) or a fragment, variant or derivative thereof, apoly(A) sequence, also called poly-A-tail, preferably at the 3′-terminusof the inventive mRNA. When present, such a poly(A) sequence comprises asequence of about 25 to about 400 adenosine nucleotides, preferably asequence of about 50 to about 400 adenosine nucleotides, more preferablya sequence of about 50 to about 300 adenosine nucleotides, even morepreferably a sequence of about 50 to about 250 adenosine nucleotides,most preferably a sequence of about 60 to about 250 adenosinenucleotides. In this context, the term “about” refers to a deviation of±10% of the value(s) it is attached to. This poly(A) sequence ispreferably located 3′ of the coding region comprised in the inventivemRNA according to the first aspect of the present invention.

According to a further preferred embodiment, the inventive mRNA can bemodified by a sequence of at least 10 cytosines, preferably at least 20cytosines, more preferably at least 30 cytosines (so-called “poly(C)sequence”). Particularly, the mRNA may contain a poly(C) sequence oftypically about 10 to 200 cytosine nucleotides, preferably about 10 to100 cytosine nucleotides, more preferably about 10 to 70 cytosinenucleotides or even more preferably about 20 to 50 or even 20 to 30cytosine nucleotides. This poly(C) sequence is preferably located 3′ ofthe coding region, more preferably 3′ of an optional poly(A) sequencecomprised in the inventive mRNA according to the first aspect of thepresent invention.

In this context, the inventive mRNA sequence may comprise in a specificembodiment:

-   -   a.) a 5′-CAP structure, preferably m7GpppN;    -   b.) a coding region encoding at least one antigenic peptide or        protein of Respiratory syncytial virus (RSV), preferably derived        from the fusion protein F of Respiratory syncytial virus (RSV);    -   c.) a poly(A) sequence preferably comprising 64 adenosines; and    -   d.) optionally, a poly(C) sequence, preferably comprising 30        cytosines.

In a particularly preferred embodiment of the first aspect of thepresent invention the inventive mRNA comprising a coding region encodingat least one antigenic peptide or protein of Respiratory syncytial virus(RSV) or a fragment, variant or derivative thereof, comprises preferablyin 5′- to 3′-direction:

-   -   a.) a 5′-CAP structure, preferably m7GpppN;    -   b.) a coding region encoding at least one antigenic peptide or        protein of Rabies virus, preferably derived from the        glycoprotein G (RAV-G) of Rabies virus;    -   c.) a poly(A) sequence preferably comprising 64 adenosines;    -   d.) optionally, a poly(C) sequence, preferably comprising 30        cytosines; and    -   e.) a histone-stem-loop, preferably comprising the corresponding        RNA sequence of the nucleic acid sequence according to SEQ ID        NO: 30.

In a further particularly preferred embodiment of the first aspect ofthe present invention, the inventive mRNA comprising a coding regionencoding at least one antigenic peptide or protein of Respiratorysyncytial virus (RSV) or a fragment, variant or derivative thereof,comprises preferably in 5′- to 3′-direction:

-   -   a.) a 5′-CAP structure, preferably m7GpppN;    -   b.) a coding region encoding at least one antigenic peptide or        protein of Respiratory syncytial virus (RSV), preferably derived        from the fusion protein F of Respiratory syncytial virus (RSV);    -   c.) optionally, a 3′-UTR element derived from an alpha globin        gene, preferably comprising the corresponding RNA sequence of        the nucleic acid sequence according to SEQ ID NO. 29, a homolog,        a fragment, or a variant thereof;    -   d.) a poly(A) sequence preferably comprising 64 adenosines;    -   e.) optionally, a poly(C) sequence, preferably comprising 30        cytosines; and    -   f.) a histone-stem-loop, preferably comprising the corresponding        RNA sequence of the nucleic acid sequence according to SEQ ID        NO: 30.

In another particular preferred embodiment, the inventive mRNA encodingat least one antigenic peptide or protein of Respiratory syncytial virus(RSV) or a fragment, variant or derivative thereof, comprises preferablyin 5′- to 3′-direction:

-   -   a.) a 5′-CAP structure, preferably m7GpppN;    -   b.) optionally, a 5′-UTR element derived from a TOP gene,        preferably derived from the corresponding RNA sequence of the        nucleic acid sequence according to SEQ ID NO. 23, a homolog, a        fragment, or a variant thereof;    -   c.) a coding region encoding at least one antigenic peptide or        protein of Respiratory syncytial virus (RSV), preferably derived        from the fusion protein F of Respiratory syncytial virus (RSV);    -   d.) optionally, a 3′UTR element derived of a gene providing a        stable mRNA, preferably derived from the corresponding RNA        sequence of a nucleic acid sequence according to SEQ ID NO. 25,        a homolog, a fragment, or a variant thereof;    -   e.) a poly(A) sequence preferably comprising 64 adenosines;    -   f.) optionally, a poly(C) sequence, preferably comprising 30        cytosines; and    -   g.) a histone-stem-loop, preferably comprising the corresponding        RNA sequence of the nucleic acid sequence according to SEQ ID        NO: 30.

The coding region might encode at least partially one of the amino acidsequences according to SEQ ID Nos. 1-11 or fragments, variants orderivatives thereof. Furthermore, the coding region of the inventivemRNA may encode a combination of at least two of these amino acidsequences or a combination of fragments, variants or derivativesthereof. Particularly preferred in this context is a combination offusion protein F with nucleoprotein N and a combination of fusionprotein F and M2-1 protein.

Additionally the coding region might be or might comprise at leastpartially one of the sequences according to SEQ ID No. 12 to SEQ ID No.22, or fragments, homologs or variants thereof. Furthermore, the mRNAmight comprise a combination of at least two of these sequences or acombination of fragments, homologs or variants thereof.

For further improvement of the resistance to e.g. in vivo degradation(e.g. by an exo- or endo-nuclease), the inventive mRNA may be providedas a stabilized nucleic acid, e.g. in the form of a modified nucleicacid. According to a further embodiment of the invention it is thereforepreferred that the inventive mRNA is stabilized, preferably by backbonemodifications, sugar modifications and/or base modifications, morepreferred stabilized by modification of the G/C-content. All of thesemodifications may be introduced into the inventive mRNA withoutimpairing the mRNA's function to be translated into the antigenicfunction derived from the Respiratory syncytial virus (RSV) peptide orprotein.

A backbone modification in the context of the present invention ispreferably a modification in which phosphates of the backbone of thenucleotides contained in the inventive mRNA are chemically modified,e.g. anionic internucleoside linkage, N3′→P5′ modifications, replacementof non-bridging oxygen atoms by boranes, neutral internucleosidelinkage, amide linkage of the nucleosides, methylene(methylimino)linkages, formacetal and thioformacetal linkages, introduction ofsulfonyl groups, or the like.

A sugar modification in the context of the present invention ispreferably a chemical modification of the sugar of the nucleotides ofthe inventive mRNA, e.g. methylation of the ribose residue or the like.

According to another embodiment, the inventive mRNA may be modified andthus stabilized by modifying the G (guanosine)/C (cytosine) content ofthe mRNA, preferably of the coding region thereof.

Therein, the G/C content of the inventive mRNA, preferably of the codingregion, is particularly increased compared to the G/C content of thecoding region of its particular wild type coding sequence, i.e. theunmodified mRNA. However, the encoded amino acid sequence of theinventive mRNA is preferably not modified compared to the coded aminoacid sequence of the particular wild type/unmodified mRNA.

The modification of the G/C-content of the inventive mRNA is based onthe fact that RNA sequences having an increased G (guanosine)/C(cytosine) content are more stable than RNA sequences having anincreased A (adenosine)/U (uracil) content. The codons of a codingsequence or a whole RNA might therefore be varied compared to the wildtype coding sequence or mRNA, such that they include an increased amountof G/C nucleotides while the translated amino acid sequence is retained.In respect to the fact that several codons code for one and the sameamino acid (so-called degeneration of the genetic code), the mostfavourable codons for the stability can be determined (so-calledalternative codon usage). Preferably, the G/C content of the codingregion of the inventive mRNA according to the invention is increased byat least 7%, more preferably by at least 15%, particularly preferably byat least 20%, compared to the G/C content of the coded region of thewild type RNA. According to a specific embodiment at least 5%, 10%, 20%,30%, 40%, 50%, 60%, more preferably at least 70%, even more preferablyat least 80% and most preferably at least 90%, 95% or even 100% of thesubstitutable codons in the region coding for a protein or peptide asdefined herein or its fragment or variant thereof or the whole sequenceof the wild type mRNA sequence or coding sequence are substituted,thereby increasing the G/C content of said sequence. In this context, itis particularly preferable to increase the G/C content of the inventivemRNA to the maximum (i.e. 100% of the substitutable codons), inparticular in the coding region, compared to the wild type sequence.

According to a further preferred embodiment of the invention, theinventive mRNA is optimized for translation, preferably optimized fortranslation by replacing codons for less frequent tRNAs of a given aminoacid by codons for more frequently occurring tRNAs of the respectiveamino acid. This is based on the finding that the translation efficiencyis also determined by a different frequency in the occurrence of tRNAsin cells. Thus, if so-called “less frequent codons” are present in theinventive mRNA to an increased extent, the corresponding modified RNA istranslated to a significantly poorer degree than in the case wherecodons coding for more frequent tRNAs are present. Preferably, thecoding region of the inventive mRNA is modified compared to thecorresponding region of the wild type RNA or coding sequence such thatat least one codon of the wild type sequence which codes for a tRNAwhich is relatively rare or less frequent in the cell is exchanged for acodon which codes for a tRNA which is more or most frequent in the celland carries the same amino acid as the relatively rare or less frequenttRNA. By this modification, the sequences of the inventive mRNA can bemodified such that codons for which more frequently occurring tRNAs areavailable are inserted. In other words, according to the invention, bythis modification all codons of the wild type sequence which code for atRNA which is relatively rare in the cell can in each case be exchangedfor a codon which codes for a respective tRNA which is relativelyfrequent in the cell and which, in each case, carries the same aminoacid as the relatively rare tRNA. Furthermore, it is particularlypreferable to link the sequential G/C content which is increased, inparticular maximized, in the inventive mRNA with the “frequent” codonswithout modifying the amino acid sequence of the protein encoded by thecoding region of the inventive mRNA or of the coding region. Thispreferred embodiment allows provision of a particularly efficientlytranslated and stabilized (modified) inventive mRNA.

Substitutions, additions or eliminations of bases are preferably carriedout using a DNA matrix for preparation of the nucleic acid molecule bytechniques of the well known site directed mutagenesis or with anoligonucleotide ligation. In such a process, for preparation of the atleast one RNA of the inventive combination vaccine as defined herein acorresponding DNA molecule may be transcribed in vitro. This DNA matrixpreferably comprises a suitable promoter, e.g. a T7 or SP6 promoter, forin vitro transcription, which is followed by the desired nucleotidesequence for the at least one RNA to be prepared and a terminationsignal for in vitro transcription. The DNA molecule, which forms thematrix of the at least one RNA of interest, may be prepared byfermentative proliferation and subsequent isolation as part of a plasmidwhich can be replicated in bacteria. Plasmids which may be mentioned assuitable for the present invention are e.g. the plasmids pT7 Ts (GenBankaccession number U26404; Lai et al., Development 1995, 121: 2349 to2360), pGEM® series, e.g. pGEM®-1 (GenBank accession number X65300; fromPromega) and pSP64 (GenBank accession number X65327); cf. also Mezei andStorts, Purification of PCR Products, in: Griffin and Griffin (ed.), PCRTechnology: Current Innovation, CRC Press, Boca Raton, FL, 2001.

In a particularly preferred embodiment, the inventive mRNA sequenceaccording to the first aspect of the present invention comprises,preferably in 5′- to 3′-direction:

-   -   a) a 5′-CAP structure, as defined herein, preferably m7GpppN;    -   b) a coding region, preferably with an increased or even        maximized G/C content compared with the G/C content of the        coding region of the wild type mRNA, encoding at least one        antigenic peptide or protein derived from the fusion protein F,        the glycoprotein G, the short hydrophobic protein SH, the matrix        protein M, the nucleoprotein N, the large polymerase L, the M2-1        protein, the M2-2 protein, the phosphoprotein P, the        non-structural protein NS1 or the non-structural protein NS2 of        Respiratory syncytial virus (RSV), or a fragment, variant or        derivative thereof;    -   c) a 3′-UTR element as defined herein, preferably derived of a        gene providing a stable mRNA, most preferably the corresponding        RNA sequence of the nucleic acid sequence according to SEQ ID        No. 29, or a homolog, a fragment or variant thereof;    -   d) a poly(A) sequence, preferably consisting of 64 adenosines    -   e) optionally a poly(C) sequence, preferably consisting of 30        cytosines.    -   f) at least one histone stem-loop sequence, preferably the        corresponding RNA sequence of the nucleic acid sequence        according to SEQ ID NO. 30.

Most preferably, the inventive mRNA sequence of that specific embodimentcomprises the sequence modifications as shown in FIG. 1 (SEQ ID NO. 31)using the example of an inventive mRNA coding for the fusion protein Fof RSV long.

In a further particularly preferred embodiment, the inventive mRNAsequence according to the first aspect of the present inventioncomprises preferably in 5′ to 3′ direction:

-   -   a) a 5′-CAP structure, as defined herein, preferably preferably        m7GpppN;    -   b) a 5′-UTR element as defined herein, preferably a 5′-UTR        element which comprises or consists of a nucleic acid sequence        which is derived from the 5′-UTR of a TOP gene, preferably the        5′-UTR of human ribosomal protein Large 32 lacking the 5′        terminal oligopyrimidine tract according to SEQ ID No. 23 or the        corresponding RNA sequence; or a fragment, homolog or variant        thereof;    -   c) a coding region, preferably with an increased or even        maximized G/C content compared with the G/C content of the        coding region of the wild type mRNA, encoding at least one        antigenic peptide or protein derived from the fusion protein F,        the glycoprotein G, the short hydrophobic protein SH, the matrix        protein M, the nucleoprotein N, the large polymerase L, the M2-1        protein, the M2-2 protein, the phosphoprotein P, the        non-structural protein NS1 or the non-structural protein NS2 of        Respiratory syncytial virus (RSV) or a fragment, variant or        derivative thereof;    -   d) a 3′-UTR element, preferably the 3′-UTR element of human        albumin according to SEQ ID No. 24 or the corresponding RNA, or        a homolog, a fragment or a variant thereof;    -   e) a poly(A) sequence, preferably consisting of 64 adenosines    -   f) optionally a poly(C) sequence, preferably consisting of 30        cytosines.    -   g) at least one histone stem-loop sequence, preferably the        corresponding RNA sequence of the nucleic acid sequence        according to SEQ ID NO. 30.

Most preferably, the inventive mRNA of that specific embodimentcomprises the sequence modifications as shown in FIG. 2 (SEQ ID NO. 32)using the example of an inventive mRNA coding for the fusion protein Fof RSV long.

In an even more particularly preferred embodiment the inventive mRNAsequence comprises or consists of the sequences shown in FIG. 1-5according to SEQ ID Nos. 31 and 35.

In further specific embodiments, the mRNA according to the invention mayfurther comprise an internal ribosome entry site (IRES) sequence orIRES-motif, which may separate several open reading frames, for exampleif the inventive mRNA encodes for two or more antigenic peptides orproteins. An IRES-sequence may be particularly helpful if the mRNA is abi- or multicistronic mRNA.

Additionally, the inventive mRNA may be prepared using any method knownin the art, including synthetic methods such as e.g. solid phasesynthesis, as well as in vitro methods, such as in vitro transcriptionreactions.

According to one embodiment of the present invention the mRNA comprisinga coding region, encoding at least one antigenic peptide or protein ofRespiratory syncytial virus (RSV) or a fragment, variant or derivativethereof may be administered naked without being associated with anyfurther vehicle, transfection or complexation agent for increasing thetransfection efficiency and/or the immunostimulatory properties of theinventive mRNA or of further comprised nucleic acid.

In a preferred embodiment, the inventive mRNA may be formulated togetherwith a cationic or polycationic compound and/or with a polymericcarrier. Accordingly, in a further embodiment of the invention it ispreferred that the inventive mRNA or any other nucleic acid comprised inthe inventive pharmaceutical composition or vaccine is associated withor complexed with a cationic or polycationic compound or a polymericcarrier, optionally in a weight ratio selected from a range of about 6:1(w/w) to about 0.25:1 (w/w), more preferably from about 5:1 (w/w) toabout 0.5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1:1(w/w) or of about 3:1 (w/w) to about 1:1 (w/w), and most preferably aratio of about 3:1 (w/w) to about 2:1 (w/w) of mRNA or nucleic acid tocationic or polycationic compound and/or with a polymeric carrier; oroptionally in a nitrogen/phosphate ratio of mRNA or nucleic acid tocationic or polycationic compound and/or polymeric carrier in the rangeof about 0.1-10, preferably in a range of about 0.3-4 or 0.3-1, and mostpreferably in a range of about 0.5-1 or 0.7-1, and even most preferablyin a range of about 0.3-0.9 or 0.5-0.9.

Thereby, the inventive mRNA or any other nucleic acid comprised in theinventive pharmaceutical composition or vaccine can also be associatedwith a vehicle, transfection or complexation agent for increasing thetransfection efficiency and/or the immunostimulatory properties of theinventive mRNA or of optionally comprised further included nucleicacids.

Cationic or polycationic compounds, being particularly preferred agentsin this context include protamine, nucleoline, spermine or spermidine,or other cationic peptides or proteins, such as poly-L-lysine (PLL),poly-arginine, basic polypeptides, cell penetrating peptides (CPPs),including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides,Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex),MAP, KALA or protein transduction domains (PTDs), PpT620, prolin-richpeptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s),Pep-1, L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides(particularly from Drosophila antennapedia), pAntp, pIs1, FGF,Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC,hCT-derived peptides, SAP, or histones.

In this context, protamine is particularly preferred.

Additionally, preferred cationic or polycationic proteins or peptidesmay be selected from the following proteins or peptides having thefollowing total formula (III):

(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x),  (formula (III))

wherein l+m+n+o+x=8-15, and l, m, n or o independently of each other maybe any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15, provided that the overall content of Arg, Lys, His and Ornrepresents at least 50% of all amino acids of the oligopeptide; and Xaamay be any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x may be anynumber selected from 0, 1, 2, 3 or 4, provided, that the overall contentof Xaa does not exceed 50% of all amino acids of the oligopeptide.Particularly preferred cationic peptides in this context are e.g. Arg₇,Arg₈, Arg₉, H₃R₉, R₉H₃, H₃R₉H₃, YSSR₉SSY, (RKH)₄, Y(RKH)₂R, etc. In thiscontext the disclosure of WO 2009/030481 is incorporated herewith byreference.

Further preferred cationic or polycationic compounds, which can be usedas transfection or complexation agent may include cationicpolysaccharides, for example chitosan, polybrene, cationic polymers,e.g. polyethyleneimine (PEI), cationic lipids, e.g. DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE:Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium,CLIPS: rac-[2 (2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, or cationic orpolycationic polymers, e.g. modified polyaminoacids, such asβ-aminoacid-polymers or reversed polyamides, etc., modifiedpolyethylenes, such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)),etc., modified acrylates, such as pDMAEMA (poly(dimethylaminoethylmethylacrylate)), etc., modified amidoamines such as pAMAM(poly(amidoamine)), etc., modified polybetaaminoester (PBAE), such asdiamine end modified 1,4 butanediol diacrylate-co-5-amino-1-pentanolpolymers, etc., dendrimers, such as polypropylamine dendrimers or pAMAMbased dendrimers, etc., polyimine(s), such as PEI: poly(ethyleneimine),poly(propyleneimine), etc., polyallylamine, sugar backbone basedpolymers, such as cyclodextrin based polymers, dextran based polymers,chitosan, etc., silan backbone based polymers, such as PMOXA-PDMScopolymers, etc., blockpolymers consisting of a combination of one ormore cationic blocks (e.g. selected from a cationic polymer as mentionedabove) and of one or more hydrophilic or hydrophobic blocks (e.g.polyethyleneglycole); etc.

A polymeric carrier used according to the invention might be a polymericcarrier formed by disulfide-crosslinked cationic components. Thedisulfide-crosslinked cationic components may be the same or differentfrom each other. The polymeric carrier can also contain furthercomponents. It is also particularly preferred that the polymeric carrierused according to the present invention comprises mixtures of cationicpeptides, proteins or polymers and optionally further components asdefined herein, which are crosslinked by disulfide bonds as describedherein. In this context, the disclosure of WO 2012/013326 isincorporated herewith by reference.

In this context, the cationic components, which form basis for thepolymeric carrier by disulfide-crosslinkage, are typically selected fromany suitable cationic or polycationic peptide, protein or polymersuitable for this purpose, particular any cationic or polycationicpeptide, protein or polymer capable to complex an mRNA or a nucleic acidas defined according to the present invention, and thereby preferablycondensing the mRNA or the nucleic acid. The cationic or polycationicpeptide, protein or polymer, is preferably a linear molecule, however,branched cationic or polycationic peptides, proteins or polymers mayalso be used.

Every disulfide-crosslinking cationic or polycationic protein, peptideor polymer of the polymeric carrier, which may be used to complex theinventive mRNA or any further nucleic acid comprised in the inventivepharmaceutical composition or vaccine contains at least one —SH moiety,most preferably at least one cysteine residue or any further chemicalgroup exhibiting an —SH moiety, capable to form a disulfide linkage uponcondensation with at least one further cationic or polycationic protein,peptide or polymer as cationic component of the polymeric carrier asmentioned herein.

As defined above, the polymeric carrier, which may be used to complexthe inventive mRNA or any further nucleic acid comprised in theinventive pharmaceutical composition or vaccine may be formed bydisulfide-crosslinked cationic (or polycationic) components.

Preferably, such cationic or polycationic peptides or proteins orpolymers of the polymeric carrier, which comprise or are additionallymodified to comprise at least one —SH moiety, are selected from,proteins, peptides and polymers as defined above for complexation agent.

In a further particular embodiment, the polymeric carrier which may beused to complex the inventive mRNA or any further nucleic acid comprisedin the inventive pharmaceutical composition or vaccine may be selectedfrom a polymeric carrier molecule according to generic formula (IV):

L-P¹—S—[S—P²—S]_(n)—S—P³-L  formula (IV)

wherein,

-   -   P¹ and P³ are different or identical to each other and represent        a linear or branched hydrophilic polymer chain, each P¹ and P³        exhibiting at least one —SH-moiety, capable to form a disulfide        linkage upon condensation with component P², or alternatively        with (AA), (AA)_(x), or [(AA)_(x)]_(z) if such components are        used as a linker between P¹ and P² or P³ and P²) and/or with        further components (e.g. (AA), (AA)_(x), [(AA)_(x)]_(z) or L),        the linear or branched hydrophilic polymer chain selected        independent from each other from polyethylene glycol (PEG),        poly-N-(2-hydroxypropyl)methacrylamide,        poly-2-(methacryloyloxy)ethyl phosphorylcholines,        poly(hydroxyalkyl L-asparagine), poly(2-(methacryloyloxy)ethyl        phosphorylcholine), hydroxyethylstarch or poly(hydroxyalkyl        L-glutamine), wherein the hydrophilic polymer chain exhibits a        molecular weight of about 1 kDa to about 100 kDa, preferably of        about 2 kDa to about 25 kDa; or more preferably of about 2 kDa        to about 10 kDa, e.g. about 5 kDa to about 25 kDa or 5 kDa to        about 10 kDa;    -   P² is a cationic or polycationic peptide or protein, e.g. as        defined above for the polymeric carrier formed by        disulfide-crosslinked cationic components, and preferably having        a length of about 3 to about 100 amino acids, more preferably        having a length of about 3 to about 50 amino acids, even more        preferably having a length of about 3 to about 25 amino acids,        e.g. a length of about 3 to 10, 5 to 15, 10 to 20 or 15 to 25        amino acids, more preferably a length of about 5 to about 20 and        even more preferably a length of about 10 to about 20; or        -   is a cationic or polycationic polymer, e.g. as defined above            for the polymeric carrier formed by disulfide-crosslinked            cationic components, typically having a molecular weight of            about 0.5 kDa to about 30 kDa, including a molecular weight            of about 1 kDa to about 20 kDa, even more preferably of            about 1.5 kDa to about 10 kDa, or having a molecular weight            of about 0.5 kDa to about 100 kDa, including a molecular            weight of about 10 kDa to about 50 kDa, even more preferably            of about 10 kDa to about 30 kDa;        -   each P² exhibiting at least two —SH-moieties, capable to            form a disulfide linkage upon condensation with further            components P² or component(s) P¹ and/or P³ or alternatively            with further components (e.g. (AA), (AA)_(x), or            [(AA)_(x)]_(z));    -   —S—S— is a (reversible) disulfide bond (the brackets are omitted        for better readability), wherein S preferably represents sulphur        or a —SH carrying moiety, which has formed a (reversible)        disulfide bond. The (reversible) disulfide bond is preferably        formed by condensation of —SH-moieties of either components P¹        and P², P² and P², or P² and P³, or optionally of further        components as defined herein (e.g. L, (AA), (AA)_(x),        [(AA)_(x)]_(z), etc); The —SH-moiety may be part of the        structure of these components or added by a modification as        defined below;    -   L is an optional ligand, which may be present or not, and may be        selected independent from the other from RGD, Transferrin,        Folate, a signal peptide or signal sequence, a localization        signal or sequence, a nuclear localization signal or sequence        (NLS), an antibody, a cell penetrating peptide, (e.g. TAT or        KALA), a ligand of a receptor (e.g. cytokines, hormones, growth        factors etc), small molecules (e.g. carbohydrates like mannose        or galactose or synthetic ligands), small molecule agonists,        inhibitors or antagonists of receptors (e.g. RGD peptidomimetic        analogues), or any further protein as defined herein, etc.;    -   n is an integer, typically selected from a range of about 1 to        50, preferably from a range of about 1, 2 or 3 to 30, more        preferably from a range of about 1, 2, 3, 4, or 5 to 25, or a        range of about 1, 2, 3, 4, or 5 to 20, or a range of about 1, 2,        3, 4, or 5 to 15, or a range of about 1, 2, 3, 4, or 5 to 10,        including e.g. a range of about 4 to 9, 4 to 10, 3 to 20, 4 to        20, 5 to 20, or 10 to 20, or a range of about 3 to 15, 4 to 15,        5 to 15, or 10 to 15, or a range of about 6 to 11 or 7 to 10.        Most preferably, n is in a range of about 1, 2, 3, 4, or 5 to        10, more preferably in a range of about 1, 2, 3, or 4 to 9, in a        range of about 1, 2, 3, or 4 to 8, or in a range of about 1, 2,        or 3 to 7.

In this context the disclosure of WO 2011/026641 is incorporatedherewith by reference. Each of hydrophilic polymers P¹ and P³ typicallyexhibits at least one —SH-moiety, wherein the at least one —SH-moiety iscapable to form a disulfide linkage upon reaction with component P² orwith component (AA) or (AA)_(x), if used as linker between P¹ and P² orP³ and P² as defined below and optionally with a further component, e.g.L and/or (AA) or (AA)_(x), e.g. if two or more —SH-moieties arecontained. The following subformulae “P¹—S—S—P²” and “P²—S—S—P³” withingeneric formula (V) above (the brackets are omitted for betterreadability), wherein any of S, P¹ and P³ are as defined herein,typically represent a situation, wherein one-SH-moiety of hydrophilicpolymers P¹ and P³ was condensed with one —SH-moiety of component P² ofgeneric formula (V) above, wherein both sulphurs of these —SH-moietiesform a disulfide bond —S—S— as defined herein in formula (V). These—SH-moieties are typically provided by each of the hydrophilic polymersP¹ and P³, e.g. via an internal cysteine or any further (modified) aminoacid or compound which carries a —SH moiety. Accordingly, thesubformulae “P¹—S—S—P²” and “P²—S—S—P³” may also be written as“P¹-Cys-Cys-P²” and “P²-Cys-Cys-P³”, if the —SH-moiety is provided by acysteine, wherein the term Cys-Cys represents two cysteines coupled viaa disulfide bond, not via a peptide bond. In this case, the term “—S—S—”in these formulae may also be written as “—S-Cys”, as “-Cys-S” or as“-Cys-Cys-”. In this context, the term “-Cys-Cys-” does not represent apeptide bond but a linkage of two cysteines via their —SH-moieties toform a disulfide bond. Accordingly, the term “-Cys-Cys-” also may beunderstood generally as “-(Cys-S)—(S-Cys)-”, wherein in this specificcase S indicates the sulphur of the —SH-moiety of cysteine. Likewise,the terms “—S-Cys” and “—Cys-S” indicate a disulfide bond between a —SHcontaining moiety and a cysteine, which may also be written as“—S—(S-Cys)” and “-(Cys-S)—S”. Alternatively, the hydrophilic polymersP¹ and P³ may be modified with a —SH moiety, preferably via a chemicalreaction with a compound carrying a —SH moiety, such that each of thehydrophilic polymers P¹ and P³ carries at least one such —SH moiety.Such a compound carrying a —SH moiety may be e.g. an (additional)cysteine or any further (modified) amino acid, which carries a —SHmoiety. Such a compound may also be any non-amino compound or moiety,which contains or allows to introduce a —SH moiety into hydrophilicpolymers P¹ and P³ as defined herein. Such non-amino compounds may beattached to the hydrophilic polymers P¹ and P³ of formula (VI) of thepolymeric carrier according to the present invention via chemicalreactions or binding of compounds, e.g. by binding of a 3-thio propionicacid or thioimolane, by amide formation (e.g. carboxylic acids,sulphonic acids, amines, etc), by Michael addition (e.g maleinimidemoieties, α,β unsatured carbonyls, etc), by click chemistry (e.g. azidesor alkines), by alkene/alkine methatesis (e.g. alkenes or alkines),imine or hydrozone formation (aldehydes or ketons, hydrazins,hydroxylamins, amines), complexation reactions (avidin, biotin, proteinG) or components which allow S_(n)-type substitution reactions (e.ghalogenalkans, thiols, alcohols, amines, hydrazines, hydrazides,sulphonic acid esters, oxyphosphonium salts) or other chemical moietieswhich can be utilized in the attachment of further components. Aparticularly preferred PEG derivate in this context isalpha-Methoxy-omega-mercapto poly(ethylene glycol). In each case, theSH-moiety, e.g. of a cysteine or of any further (modified) amino acid orcompound, may be present at the terminal ends or internally at anyposition of hydrophilic polymers P¹ and P³. As defined herein, each ofhydrophilic polymers P¹ and P³ typically exhibits at least one —SH—moiety preferably at one terminal end, but may also contain two or evenmore —SH-moieties, which may be used to additionally attach furthercomponents as defined herein, preferably further functional peptides orproteins e.g. a ligand, an amino acid component (AA) or (AA)_(x),antibodies, cell penetrating peptides or enhancer peptides (e.g. TAT,KALA), etc.

In this context, it is particularly preferred that the inventive mRNA iscomplexed at least partially with a cationic or polycationic compoundand/or a polymeric carrier, preferably cationic proteins or peptides. Inthis context the disclosure of WO 2010/037539 and WO 2012/113513 isincorporated herewith by reference. Partially means that only a part ofthe inventive mRNA is complexed with a cationic compound and that therest of the inventive mRNA is (comprised in the inventive pharmaceuticalcomposition or vaccine) in uncomplexed form (“free”). Preferably theratio of complexed mRNA to:free mRNA (in the inventive pharmaceuticalcomposition or vaccine) is selected from a range of about 5:1 (w/w) toabout 1:10 (w/w), more preferably from a range of about 4:1 (w/w) toabout 1:8 (w/w), even more preferably from a range of about 3:1 (w/w) toabout 1:5 (w/w) or 1:3 (w/w), and most preferably the ratio of complexedmRNA to free mRNA in the inventive pharmaceutical composition or vaccineis selected from a ratio of about 1:1 (w/w).

The complexed mRNA in the inventive pharmaceutical composition orvaccine, is preferably prepared according to a first step by complexingthe inventive mRNA with a cationic or polycationic compound and/or witha polymeric carrier, preferably as defined herein, in a specific ratioto form a stable complex. In this context, it is highly preferable, thatno free cationic or polycationic compound or polymeric carrier or only anegligibly small amount thereof remains in the component of thecomplexed mRNA after complexing the mRNA. Accordingly, the ratio of themRNA and the cationic or polycationic compound and/or the polymericcarrier in the component of the complexed mRNA is typically selected ina range that the mRNA is entirely complexed and no free cationic orpolycationic compound or polymeric carrier or only a negligibly smallamount thereof remains in the composition.

Preferably the ratio of the mRNA to the cationic or polycationiccompound and/or the polymeric carrier, preferably as defined herein, isselected from a range of about 6:1 (w/w) to about 0.25:1 (w/w), morepreferably from about 5:1 (w/w) to about 0.5:1 (w/w), even morepreferably of about 4:1 (w/w) to about 1:1 (w/w) or of about 3:1 (w/w)to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w) toabout 2:1 (w/w). Alternatively, the ratio of the mRNA to the cationic orpolycationic compound and/or the polymeric carrier, preferably asdefined herein, in the component of the complexed mRNA, may also becalculated on the basis of the nitrogen/phosphate ratio (N/P-ratio) ofthe entire complex. In the context of the present invention, anN/P-ratio is preferably in the range of about 0.1-10, preferably in arange of about 0.3-4 and most preferably in a range of about 0.5-2 or0.7-2 regarding the ratio of mRNA:cationic or polycationic compoundand/or polymeric carrier, preferably as defined herein, in the complex,and most preferably in a range of about 0.7-1.5, 0.5-1 or 0.7-1, andeven most preferably in a range of about 0.3-0.9 or 0.5-0.9, preferablyprovided that the cationic or polycationic compound in the complex is acationic or polycationic cationic or polycationic protein or peptideand/or the polymeric carrier as defined above. In this specificembodiment the complexed mRNA is also emcompassed in the term “adjuvantcomponent”.

In a further aspect, the invention provides for a composition comprisinga plurality or more than one, preferably 2 to 10, more preferably 2 to5, most preferably 2 to 4 of the inventive mRNA sequences as definedherein. These inventive compositions comprise more than one inventivemRNA sequences, preferably encoding different peptides or proteins whichcomprise preferably different pathogenic antigens or fragments, variantsor derivatives thereof. Particularly preferred in this context is thatat least one mRNA sequence encodes at least one antigenic peptide orprotein derived from the fusion protein F of Respiratory syncytial virus(RSV) and that at least one mRNA sequence encodes at least one antigenicpeptide or protein derived from another antigen of Respiratory syncytialvirus (RSV), particularly of nucleoprotein N or M2-1 protein. Furtherparticularly preferred combinations of antigens are in this context:

-   -   F+G (serotype A)+G (serotype B)    -   F+G (serotype A)+G (serotype B)+M2−1    -   F+G (serotype A)+G (serotype B)+N    -   F+G (serotype A)+G (serotype B)+N+M2−1    -   F+M2−1+N    -   F+M2−1    -   F+N    -   F+G (serotype A)+G (serotype B)+N+M2−1+P+M2−2+M+L    -   F+G (serotype A)+G (serotype B)+N+M2−1+P+M2−2+M    -   F+G (serotype A)+G (serotype B)+N+M2−1+P+M2−2+L    -   F+G (serotype A)+G (serotype B)+N+M2−1+P+M+L    -   F+G (serotype A)+G (serotype B)+N+M2−1+M2−2+M+L    -   F+G (serotype A)+G (serotype B)+N+P+M2−2+M+L    -   F+G (serotype A)+G (serotype B)+M2−1+P+M2−2+M+L

Accordingly, in a further particular preferred aspect, the presentinvention also provides a pharmaceutical composition, comprising atleast one inventive mRNA sequence as defined herein or an inventivecomposition comprising a plurality of inventive mRNA sequences asdefined herein and optionally a pharmaceutically acceptable carrierand/or vehicle.

As a first ingredient, the inventive pharmaceutical compositioncomprises at least one inventive mRNA sequence as defined herein.

As a second ingredient, the inventive pharmaceutical composition mayoptionally comprise at least one additional pharmaceutically activecomponent. A pharmaceutically active component in this connection is acompound that has a therapeutic effect to heal, ameliorate or prevent aparticular indication or disease as mentioned herein, preferably RSVinfections. Such compounds include, without implying any limitation,peptides or proteins, preferably as defined herein, nucleic acids,preferably as defined herein, (therapeutically active) low molecularweight organic or inorganic compounds (molecular weight less than 5000,preferably less than 1000), sugars, antigens or antibodies, preferablyas defined herein, therapeutic agents already known in the prior art,antigenic cells, antigenic cellular fragments, cellular fractions; cellwall components (e.g. polysaccharides), modified, attenuated orde-activated (e.g. chemically or by irradiation) pathogens (virus,bacteria etc.), adjuvants, preferably as defined herein, etc.Particularly preferred in this context are RSV vaccines, or RSV immuneglobulines, e.g. Palivizumab (Synagis®).

The inventive pharmaceutical composition may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term parenteralas used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional, intracranial, transdermal, intradermal,intrapulmonal, intraperitoneal, intracardial, intraarterial, andsublingual injection or infusion techniques.

Particularly preferred is intradermal and intramuscular injection.Sterile injectable forms of the inventive pharmaceutical compositionsmay be aqueous or oleaginous suspension. These suspensions may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents.

In a preferred embodiment, the inventive pharmaceutical composition isadministered via intradermal or intramuscular injection, preferably byusing conventional needle-based injection technique or by using aneedle-free system, e.g. jet injection. In a further preferredembodiment, the inventive pharmaceutical composition may be administeredby jet injection as defined herein. Preferably, the inventivepharmaceutical composition may be adiminstered intramuscularly by jetinjection. According to another embodiment, the pharmaceuticalcomposition is administered intradermally via jet injection.

In a preferred embodiment, the pharmaceutical composition may beadministered once, twice or three times, preferably by intradermal orintramuscular injection, preferably by jet injection. According to acertain embodiment, a single administration of the inventivepharmaceutical composition, preferably via intradermal or intramuscularinjection, preferably by using jet injection, is sufficient foreliciting an immune response against the at least one antigen encoded bythe mRNA sequence according to the invention. In a preferred embodiment,the single administration of the pharmaceutical composition elicits animmune response resulting in virus neutralisation. In this context, onesingle intradermal or intramuscular injection of the pharmaceuticalcomposition is particularly preferred. Preferably, furtheradministrations of the pharmaceutical composition may optionally becarried out in order to enhance and/or prolong the immune response.

According to a specific embodiment, the inventive pharmaceuticalcomposition may comprise an adjuvant. In this context, an adjuvant maybe understood as any compound, which is suitable to initiate or increasean immune response of the innate immune system, i.e. a non-specificimmune response. With other words, when administered, the inventivepharmaceutical composition preferably elicits an innate immune responsedue to the adjuvant, optionally contained therein. Preferably, such anadjuvant may be selected from an adjuvant known to a skilled person andsuitable for the present case, i.e. supporting the induction of aninnate immune response in a mammal, e.g. an adjuvant protein as definedabove or an adjuvant as defined in the following.

Particularly preferred as adjuvants suitable for depot and delivery arecationic or polycationic compounds as defined above for the inventivemRNA sequence as vehicle, transfection or complexation agent.

Furthermore, the inventive pharmaceutical composition may comprise oneor more additional adjuvants, which are suitable to initiate or increasean immune response of the innate immune system, i.e. a non-specificimmune response, particularly by binding to pathogen-associatedmolecular patterns (PAMPs). With other words, when administered, thepharmaceutical composition or vaccine preferably elicits an innateimmune response due to the adjuvant, optionally contained therein.Preferably, such an adjuvant may be selected from an adjuvant known to askilled person and suitable for the present case, i.e. supporting theinduction of an innate immune response in a mammal, e.g. an adjuvantprotein as defined above or an adjuvant as defined in the following.According to one embodiment such an adjuvant may be selected from anadjuvant as defined above.

Also such an adjuvant may be selected from any adjuvant known to askilled person and suitable for the present case, i.e. supporting theinduction of an innate immune response in a mammal and/or suitable fordepot and delivery of the components of the inventive pharmaceuticalcomposition or vaccine. Preferred as adjuvants suitable for depot anddelivery are cationic or polycationic compounds as defined above.Likewise, the adjuvant may be selected from the group consisting of,e.g., cationic or polycationic compounds as defined above, fromchitosan, TDM, MDP, muramyl dipeptide, pluronics, alum solution,aluminium hydroxide, ADJUMER™ (polyphosphazene); aluminium phosphategel; glucans from algae; algammulin; aluminium hydroxide gel (alum);highly protein-adsorbing aluminium hydroxide gel; low viscosityaluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80(0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4);AVRIDINE™ (propanediamine); BAY R1005™((N-(2-deoxy-2-L-leucylaminob-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amidehydroacetate); CALCITRIOL™ (1-alpha,25-dihydroxy-vitamin D3); calciumphosphate gel; CAP™ (calcium phosphate nanoparticles); choleraholotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein,sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205);cytokine-containing liposomes; DDA (dimethyldioctadecylammoniumbromide); DHEA (dehydroepiandrosterone); DMPC(dimyristoylphosphatidylcholine); DMPG(dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acidsodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant;gamma inulin; Gerbu adjuvant (mixture of: i)N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D35 glutamine(GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii)zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP(N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L47 alanyl-D-isoglutamine);imiquimod (1-(2-methypropyl)-1H-imidazo[4,5-c]quinoline-4-amine);ImmTher™(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmitate); DRVs (immunoliposomes prepared fromdehydration-rehydration vesicles); interferongamma; interleukin-1beta;interleukin-2; interleukin-7; interleukin-12; ISCOMS™; ISCOPREP7.0.3.™;liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT 5 oral adjuvant (E.coli labile enterotoxin-protoxin); microspheres and microparticles ofany composition; MF59™; (squalenewater emulsion); MONTANIDE ISA 51™(purified incomplete Freund's adjuvant); MONTANIDE ISA 720™(metabolisable oil adjuvant); MPL™ (3-Q-desacyl-4′-monophosphoryl lipidA); MTP-PE and MTP-PE liposomes((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide,monosodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE™and DMURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl);NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles ofany composition; NISVs (non-ionic surfactant vesicles); PLEURAN™(□β-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid andglycolic acid; microspheres/nanospheres); PLURONIC L121™; PMMA(polymethylmethacrylate); PODDS™ (proteinoid microspheres); polyethylenecarbamate derivatives; poly-rA: poly-rU (polyadenylic acid-polyuridylicacid complex); poly sorbate 80 (Tween 80); protein cochleates (AvantiPolar Lipids, Inc., Alabaster, AL); STIMULON™ (QS-21); Quil-A (Quil-Asaponin); S-28463(4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol);SAF-1™ (“Syntex adjuvant formulation”); Sendai proteoliposomes andSendai containing lipid matrices; Span-85 (sorbitan trioleate); Specol(emulsion of Marcol 52, Span 85 and Tween 85); squalene or Robane®(2,6,10,15,19,23-hexamethyltetracosan and2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane); stearyltyrosine (octadecyltyro sine hydrochloride); Theramid®(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Aladipalmitoxypropylamide);Theronyl-MDP (Termurtide™ or [thr 1]-MDP;N-acetylmuramyl-Lthreonyl-D-isoglutamine); Ty particles (Ty-VLPs orvirus-like particles); Walter-Reed liposomes (liposomes containing lipidA adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys,in particular aluminium salts, such as Adju-phos, Alhydrogel,Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax,Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121,Poloaxmer4010), etc.; liposomes, including Stealth, cochleates,including BIORAL; plant derived adjuvants, including QS21, Quil A,Iscomatrix, ISCOM; adjuvants suitable for costimulation includingTomatine, biopolymers, including PLG, PMM, Inulin, microbe derivedadjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleicacid sequences, CpG7909, ligands of human TLR 1-10, ligands of murineTLR 1-13, ISS-1018, 35 IC31, Imidazoquinolines, Ampligen, Ribi529,IMOxine, IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys,Flagellin, GPI anchor, LNFPIII/Lewis X, antimicrobial peptides,UC-1V150, RSV fusion protein, cdiGMP; and adjuvants suitable asantagonists including CGRP neuropeptide.

Particularly preferred, an adjuvant may be selected from adjuvants,which support induction of a Th1-immune response or maturation of naïveT-cells, such as GM-CSF, IL-12, IFNg, any immunostimulatory nucleic acidas defined above, preferably an immunostimulatory RNA, CpG DNA, etc.

In a further preferred embodiment, it is also possible that theinventive pharmaceutical composition contains besides theantigen-providing mRNA further components, which are selected from thegroup comprising: further antigens or further antigen-providing nucleicacids; a further immunotherapeutic agent; one or more auxiliarysubstances; or any further compound, which is known to beimmunostimulating due to its binding affinity (as ligands) to humanToll-like receptors; and/or an adjuvant nucleic acid, preferably animmunostimulatory RNA (isRNA).

The inventive pharmaceutical composition can additionally contain one ormore auxiliary substances in order to increase its immunogenicity orimmunostimulatory capacity, if desired. A synergistic action of theinventive mRNA sequence as defined herein and of an auxiliary substance,which may be optionally contained in the inventive pharmaceuticalcomposition, is preferably achieved thereby. Depending on the varioustypes of auxiliary substances, various mechanisms can come intoconsideration in this respect. For example, compounds that permit thematuration of dendritic cells (DCs), for example lipopolysaccharides,TNF-alpha or CD40 ligand, form a first class of suitable auxiliarysubstances. In general, it is possible to use as auxiliary substance anyagent that influences the immune system in the manner of a “dangersignal” (LPS, GP96, etc.) or cytokines, such as GM-CFS, which allow animmune response to be enhanced and/or influenced in a targeted manner.Particularly preferred auxiliary substances are cytokines, such asmonokines, lymphokines, interleukins or chemokines, that further promotethe innate immune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27,IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta,IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors,such as hGH.

Further additives, which may be included in the inventive pharmaceuticalcomposition, are emulsifiers, such as, for example, Tween®; wettingagents, such as, for example, sodium lauryl sulfate; colouring agents;taste-imparting agents, pharmaceutical carriers; tablet-forming agents;stabilizers; antioxidants; preservatives.

The inventive pharmaceutical composition can also additionally containany further compound, which is known to be immunostimulating due to itsbinding affinity (as ligands) to human Toll-like receptors TLR1, TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its bindingaffinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

In this context, it is particularly preferred that the optionallycomprised adjuvant component comprises the same inventive mRNA ascomprised in the inventive pharmaceutical composition asantigen-providing mRNA e.g. mRNA coding for an antigenic peptide orprotein of RSV infections Respiratory syncytial virus (RSV) orfragments, variants or derivatives thereof.

Despite, the inventive pharmaceutical composition may comprise furthercomponents for facilitating administration and uptake of components ofthe pharmaceutical composition. Such further components may be anappropriate carrier or vehicle, additional adjuvants for supporting anyimmune response, antibacterial and/or antiviral agents.

Accordingly, in a further embodiment, the inventive pharmaceuticalcomposition furthermore comprises a pharmaceutically acceptable carrierand/or vehicle.

Such a pharmaceutically acceptable carrier typically includes the liquidor non-liquid basis of a composition comprising the components of theinventive pharmaceutical composition. If the composition is provided inliquid form, the carrier will typically be pyrogen-free water; isotonicsaline or buffered (aqueous) solutions, e.g. phosphate, citrate etc.buffered solutions. The injection buffer may be hypertonic, isotonic orhypotonic with reference to the specific reference medium, i.e. thebuffer may have a higher, identical or lower salt content with referenceto the specific reference medium, wherein preferably such concentrationsof the afore mentioned salts may be used, which do not lead to damage ofcells due to osmosis or other concentration effects. Reference media aree.g. liquids occurring in “in vivo” methods, such as blood, lymph,cytosolic liquids, or other body liquids, or e.g. liquids, which may beused as reference media in “in vitro” methods, such as common buffers orliquids. Such common buffers or liquids are known to a skilled person.Ringer-Lactate solution is particularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds, which are suitable for administration to apatient to be treated, may be used as well for the pharmaceuticalcomposition according to the invention. The term “compatible” as usedhere means that these constituents of the inventive pharmaceuticalcomposition are capable of being mixed with the components of theinventive pharmaceutical composition in such a manner that nointeraction occurs which would substantially reduce the pharmaceuticaleffectiveness of the pharmaceutical composition under typical useconditions.

A further component of the inventive pharmaceutical composition may bean immunotherapeutic agent that can be selected from immunoglobulins,preferably IgGs, monoclonal or polyclonal antibodies, polyclonal serumor sera, etc, most preferably immunoglobulins directed againstRespiratory syncytial virus (RSV), e.g. Palivizumab. Preferably, such afurther immunotherapeutic agent may be provided as a peptide/protein ormay be encoded by a nucleic acid, preferably by a DNA or an RNA, morepreferably an mRNA. Such an immunotherapeutic agent allows providingpassive vaccination additional to active vaccination triggered by theinventive antigen-providing mRNA.

Furthermore, in a specific embodiment, additionally to theantigen-providing mRNA further antigens can be included in the inventivepharmaceutical composition and are typically substances such as cells,cell lysates, viruses, attenuated viruses, inactivated viruses,proteins, peptides, nucleic acids or other bio- or macromolecules orfragments thereof. Preferably, antigens may be proteins and peptides orfragments thereof, such as epitopes of those proteins or peptides,preferably having 5 to 15, more preferably 6 to 9, amino acids.Particularly, said proteins, peptides or epitopes may be derived fromthe fusion protein F, the glycoprotein G, the short hydrophobic proteinSH, the matrix protein M, the nucleoprotein N, the large polymerase L,the M2-1 protein, the M2-2 protein, the phosphoprotein P, thenon-structural protein NS1 or the non-structural protein NS2 ofRespiratory syncytial virus (RSV), or from fragments, variants orderivatives thereof. Further, antigens may also comprise any otherbiomolecule, e.g., lipids, carbohydrates, etc. Preferably, the antigenis a protein or (poly-) peptide antigen, a nucleic acid, a nucleic acidencoding a protein or (poly-) peptide antigen, a polysaccharide antigen,a polysaccharide conjugate antigen, a lipid antigen, a glycolipidantigen, a carbohydrate antigen, a bacterium, a cell (vaccine), orkilled or attenuated viruses.

The inventive pharmaceutical composition or vaccine as defined hereinmay furthermore comprise further additives or additional compounds.Further additives, which may be included in the pharmaceuticalcomposition, are emulsifiers, such as, for example, Tween®; wettingagents, such as, for example, sodium lauryl sulfate; colouring agents;taste-imparting agents, pharmaceutical carriers; tablet-forming agents;stabilizers; antioxidants; preservatives, RNase inhibitors and/or ananti-bacterial agent or an anti-viral agent. Additionally the inventivepharmaceutical composition may comprise small interfering RNA (siRNA)directed against genes of Respiratory syncytial virus (RSV), e.g. siRNAdirected against the gene encoding the fusion protein F, theglycoprotein G, the short hydrophobic protein SH, the matrix protein M,the nucleoprotein N, the large polymerase L, the M2-1 protein, the M2-2protein, the phosphoprotein P, the non-structural protein NS1 or thenon-structural protein NS2 of Respiratory syncytial virus (RSV). Theinventive pharmaceutical composition typically comprises a “safe andeffective amount” of the components of the inventive pharmaceuticalcomposition, particularly of the inventive mRNA sequence(s) as definedherein. As used herein, a “safe and effective amount” means an amount ofthe inventive mRNA sequence(s) as defined herein as such that issufficient to significantly induce a positive modification of a diseaseor disorder or to prevent a disease, preferably RSV infections asdefined herein. At the same time, however, a “safe and effective amount”is small enough to avoid serious side-effects and to permit a sensiblerelationship between advantage and risk. The determination of theselimits typically lies within the scope of sensible medical judgment.

The inventive pharmaceutical composition may be used for human and alsofor veterinary medical purposes, preferably for human medical purposes,as a pharmaceutical composition in general or as a vaccine.

According to another particularly preferred aspect, the inventivepharmaceutical composition (or the inventive mRNA sequence as definedherein or the inventive composition comprising a plurality of inventivemRNA sequences as defined herein) may be provided or used as a vaccine.Typically, such a vaccine is as defined above for pharmaceuticalcompositions. Additionally, such a vaccine typically contains theinventive mRNA sequence as defined herein or the inventive compositioncomprising a plurality of inventive mRNA sequences as defined herein.

The inventive vaccine may also comprise a pharmaceutically acceptablecarrier, adjuvant, and/or vehicle as defined herein for the inventivepharmaceutical composition. In the specific context of the inventivevaccine, the choice of a pharmaceutically acceptable carrier isdetermined in principle by the manner in which the inventive vaccine isadministered. The inventive vaccine can be administered, for example,systemically or locally. Routes for systemic administration in generalinclude, for example, transdermal, oral, parenteral routes, includingsubcutaneous, intravenous, intramuscular, intraarterial, intradermal andintraperitoneal injections and/or intranasal administration routes.Routes for local administration in general include, for example, topicaladministration routes but also intradermal, transdermal, subcutaneous,or intramuscular injections or intralesional, intracranial,intrapulmonal, intracardial, and sublingual injections. More preferably,vaccines may be administered by an intradermal, subcutaneous, orintramuscular route. Inventive vaccines are therefore preferablyformulated in liquid (or sometimes in solid) form.

In a preferred embodiment, the inventive vaccine is administered viaintradermal or intramuscular injection, preferably by using conventionalneedle-based injection technique or by using a needle-free system, e.g.jet injection. In a further preferred embodiment, the inventive vaccinemay be administered by jet injection as defined herein. Preferably, theinventive vaccine is administered intramuscularly by jet injection.According to another embodiment, the vaccine is administeredintradermally via jet injection.

In a preferred embodiment, the vaccine may be administered once, twiceor three times, preferably by intradermal or intramuscular injection,preferably by jet injection. According to a certain embodiment, a singleadministration of the inventive vaccine, preferably via intradermal orintramuscular injection, preferably by using jet injection, issufficient for eliciting an immune response against the at least oneantigen encoded by the mRNA sequence according to the invention. In apreferred embodiment, the single administration of the vaccine elicitsan immune response resulting in virus neutralisation. In this context,one single intradermal or intramuscular injection of the vaccine isparticularly preferred. Preferably, further administrations of thevaccine may optionally be carried out in order to enhance and/or prolongthe immune response.

The inventive vaccine can additionally contain one or more auxiliarysubstances in order to increase its immunogenicity or immunostimulatorycapacity, if desired. Particularly preferred are adjuvants as auxiliarysubstances or additives as defined for the pharmaceutical composition.

In a further aspect, the invention is directed to a kit or kit of partscomprising the components of the inventive mRNA sequence, the inventivecomposition comprising a plurality of inventive mRNA sequences, theinventive pharmaceutical composition or vaccine and optionally technicalinstructions with information on the administration and dosage of thecomponents.

Beside the components of the inventive mRNA sequence, the inventivecomposition comprising a plurality of inventive mRNA sequences, theinventive pharmaceutical composition or vaccine the kit may additionallycontain a pharmaceutically acceptable vehicle, an adjuvant and at leastone further component as defined herein, as well as means foradministration and technical instructions. The components of theinventive mRNA sequence, the inventive composition comprising aplurality of inventive mRNA sequences, the inventive pharmaceuticalcomposition or vaccine and e.g. the adjuvant may be provided inlyophilized form. In a preferred embodiment, prior to use of the kit forvaccination, the provided vehicle is than added to the lyophilizedcomponents in a predetermined amount as written e.g. in the providedtechnical instructions. By doing so the inventive mRNA sequence, theinventive composition comprising a plurality of inventive mRNAsequences, the inventive pharmaceutical composition or vaccine,according to the above described aspects of the invention is providedthat can afterwards be used in a method as described above, also.

The present invention furthermore provides several applications and usesof the inventive mRNA sequence as defined herein, of the inventivecomposition comprising a plurality of inventive mRNA sequences asdefined herein, of the inventive pharmaceutical composition, of theinventive vaccine, all comprising the inventive mRNA sequence as definedherein or of kits comprising same.

In a further aspect, the invention provides an mRNA sequence encoding atleast one antigenic peptide or protein of Respiratory syncytial virus(RSV), or a fragment, variant or derivative thereof, and a composition,a pharmaceutical composition, a vaccine and a kit, all comprising themRNA sequence for use in a method of prophylactic and/or therapeutictreatment of RSV infections. Consequently, in a further aspect, thepresent invention is directed to the first medical use of the inventivemRNA sequence, the inventive composition comprising a plurality ofinventive mRNA sequences, the inventive pharmaceutical composition, theinventive vaccine, and the inventive kit as defined herein as amedicament. Particularly, the invention provides the use of an mRNAsequence encoding at least one antigenic peptide or protein ofRespiratory syncytial virus (RSV), or a fragment, variant or derivativethereof as defined above for the preparation of a medicament.

According to another aspect, the present invention is directed to thesecond medical use of the mRNA sequence encoding at least one antigenicpeptide or protein of Respiratory syncytial virus (RSV), or a fragment,variant or derivative thereof, as defined herein, optionally in form ofa composition comprising a plurality of inventive mRNA sequences, apharmaceutical composition or vaccine, kit or kit of parts, for thetreatment of RSV infections as defined herein. Particularly, the mRNAsequence encoding at least one antigenic peptide or protein ofRespiratory syncytial virus (RSV), or a fragment, variant or derivativethereof to be used in a method as said above is a mRNA sequenceformulated together with a pharmaceutically acceptable vehicle and anoptionally additional adjuvant and an optionally additional furthercomponent as defined above e.g. a further antigen or a RSV immuneglobuline. In this context particularly the (prophylactic) treatment ofinfants, the elderly and immunocompromised patients is preferred. Andeven more preferred is the (prophylactic) treatment of pre-term infantsand infants with chronic lung disease.

The inventive mRNA sequence may alternatively be provided such that itis administered for preventing or treating RSV infections by severaldoses, each dose containing the inventive mRNA sequence encoding atleast one antigenic peptide or protein of RSV infections Respiratorysyncytial virus (RSV), or a fragment, variant or derivative thereof,e.g. the first dose containing at least one mRNA encoding at least oneantigenic peptide or protein derived from the fusion protein F (orfragments, variants or derivatives thereof) and the second dosecontaining at least one mRNA sequence encoding at least one antigenicpeptide or protein derived from a different antigen of Respiratorysyncytial virus (RSV), preferably from the nucleoprotein N (orfragments, variants or derivatives thereof), from the M2-1 protein orthe glycoprotein G (or fragments, variants or derivatives thereof). Bythat embodiment, both doses are administered in a staggered way, i.e.subsequently, shortly one after the other, e.g. within less than 10minutes, preferably less than 2 minutes, and at the same site of thebody to achieve the same immunological effect as for administration ofone single composition containing both, e.g. the mRNA encoding thefusion protein F and the mRNA encoding the nucleoprotein N.

According to a specific embodiment, the inventive mRNA sequence, or theinventive pharmaceutical composition or vaccine may be administered tothe patient as a single dose. In certain embodiments, the inventive mRNAsequence or the inventive pharmaceutical composition or vaccine may beadministered to a patient as a single dose followed by a second doselater and optionally even a third, fourth (or more) dose subsequentthereto etc. In accordance with this embodiment, booster inoculationswith the inventive mRNA sequence or the inventive pharmaceuticalcomposition or vaccine may be administered to a patient at specific timeintervals, preferably as defined below, following the second (or third,fourth, etc.) inoculation. In this context, it is particularly preferredthat several doses comprise the same mRNA sequence encoding the sameantigenic peptide or protein of Respiratory syncytial virus (RSV), e.g.fusion protein F. In that embodiment the doses are given in a specifictime period e.g. 20-30 days. For example for post-exposure prophylaxisat least 5 doses of the inventive mRNA sequence or inventivepharmaceutical composition or vaccine can be administered in 20-30 days.

In a preferred embodiment, inventive mRNA sequence, inventivepharmaceutical composition or vaccine is administered via intradermal orintramuscular injection, preferably by using conventional needle-basedinjection technique or by using a needle-free system, e.g. jetinjection. In a further preferred embodiment, the inventive mRNAsequence, inventive pharmaceutical composition or vaccine may beadministered by jet injection as defined herein. Preferably, theinventive mRNA sequence, inventive pharmaceutical composition or vaccineis adiminstered intramuscularly by jet injection. According to anotherembodiment, the inventive mRNA sequence, inventive pharmaceuticalcomposition or vaccine is administered intradermally via jet injection.

In a preferred embodiment, the inventive mRNA sequence, inventivepharmaceutical composition or vaccine may be administered once, twice orthree times, preferably by intradermal or intramuscular injection,preferably by jet injection. According to a certain embodiment, a singleadministration of the the inventive mRNA sequence, inventivepharmaceutical composition or vaccine, preferably via intradermal orintramuscular injection, preferably by using jet injection, issufficient for eliciting an immune response against the at least oneantigen encoded by the mRNA sequence according to the invention. In apreferred embodiment, the single administration of the inventive mRNAsequence, inventive pharmaceutical composition or vaccine elicits animmune response resulting in virus neutralisation. In this context, onesingle intradermal or intramuscular injection of the inventive mRNAsequence, inventive pharmaceutical composition or vaccine isparticularly preferred. Preferably, further administrations of theinventive mRNA sequence, inventive pharmaceutical composition or vaccinemay optionally be carried out in order to enhance and/or prolong theimmune response.

In certain embodiments, such booster inoculations with the inventivemRNA sequence or inventive pharmaceutical composition or vaccine mayutilize an additional compound or component as defined for the inventivemRNA sequence or inventive pharmaceutical composition or vaccine asdefined herein.

According to a further aspect, the present invention also provides amethod for expression of an encoded antigenic peptide or protein derivedfrom the fusion protein F, the glycoprotein G, the short hydrophobicprotein SH, the matrix protein M, the nucleoprotein N, the largepolymerase L, the M2-1 protein, the M2-2 protein, the phosphoprotein P,the non-structural protein NS1 or the non-structural protein NS2 ofRespiratory syncytial virus (RSV) comprising the steps, e.g. a)providing the inventive mRNA sequence as defined herein or the inventivecomposition comprising a plurality of inventive mRNA sequences asdefined herein, b) applying or administering the inventive mRNA sequenceas defined herein or the inventive composition comprising a plurality ofinventive mRNA sequences as defined herein to an expression system, e.g.to a cell-free expression system, a cell (e.g. an expression host cellor a somatic cell), a tissue or an organism. The method may be appliedfor laboratory, for research, for diagnostic, for commercial productionof peptides or proteins and/or for therapeutic purposes. In thiscontext, typically after preparing the inventive mRNA sequence asdefined herein or of the inventive composition comprising a plurality ofinventive mRNA sequences as defined herein, it is typically applied oradministered to a cell-free expression system, a cell (e.g. anexpression host cell or a somatic cell), a tissue or an organism, e.g.in naked or complexed form or as a pharmaceutical composition or vaccineas described herein, preferably via transfection or by using any of theadministration modes as described herein. The method may be carried outin vitro, in vivo or ex vivo. The method may furthermore be carried outin the context of the treatment of a specific disease, particularly inthe treatment of infectious diseases, preferably RSV infections asdefined herein.

In this context, in vitro is defined herein as transfection ortransduction of the inventive mRNA as defined herein or of the inventivecomposition comprising a plurality of inventive mRNA sequences asdefined herein into cells in culture outside of an organism; in vivo isdefined herein as transfection or transduction of the inventive mRNA orof the inventive composition comprising a plurality of inventive mRNAsequences into cells by application of the inventive mRNA or of theinventive composition to the whole organism or individual and ex vivo isdefined herein as transfection or transduction of the inventive mRNA orof the inventive composition comprising a plurality of inventive mRNAsequences into cells outside of an organism or individual and subsequentapplication of the transfected cells to the organism or individual.

Likewise, according to another aspect, the present invention alsoprovides the use of the inventive mRNA sequence as defined herein or ofthe inventive composition comprising a plurality of inventive mRNAsequences as defined herein, preferably for diagnostic or therapeuticpurposes, for expression of an encoded antigenic peptide or protein,e.g. by applying or administering the inventive mRNA sequence as definedherein or of the inventive composition comprising a plurality ofinventive mRNA sequences as defined herein, e.g. to a cell-freeexpression system, a cell (e.g. an expression host cell or a somaticcell), a tissue or an organism. The use may be applied for laboratory,for research, for diagnostic for commercial production of peptides orproteins and/or for therapeutic purposes. In this context, typicallyafter preparing the inventive mRNA sequence as defined herein or of theinventive composition comprising a plurality of inventive mRNA sequencesas defined herein, it is typically applied or administered to acell-free expression system, a cell (e.g. an expression host cell or asomatic cell), a tissue or an organism, preferably in naked form orcomplexed form, or as a pharmaceutical composition or vaccine asdescribed herein, preferably via transfection or by using any of theadministration modes as described herein. The use may be carried out invitro, in vivo or ex vivo. The use may furthermore be carried out in thecontext of the treatment of a specific disease, particularly in thetreatment of RSV infections.

In a further aspect, the invention provides a method of treatment orprophlaxis of RSV infections comprising the steps:

-   -   a) providing the inventive mRNA sequence, the composition        comprising a plurality of inventive mRNA sequences, the        pharmaceutical composition or the kit or kit of parts comprising        the inventive mRNA sequence as defined above;    -   b) applying or administering the mRNA sequence, the composition,        the pharmaceutical composition or the kit or kit of parts to a        tissue or an organism;    -   c) optionally administering RSV immune globuline.

Taken together, the invention provides in a certain aspect an mRNAsequence comprising a coding region encoding at least one antigenicpeptide or protein of Respiratory syncytial virus (RSV). The inventivemRNA sequence is for use in a method of prophylactic and/or therapeutictreatment of infections caused by syncytial virus (RSV). Accordingly,the invention relates to an mRNA sequence as defined herein for use in amethod of prophylactic and/or therapeutic treatment of RSV infections.

In the present invention, if not otherwise indicated, different featuresof alternatives and embodiments may be combined with each other, wheresuitable. Furthermore, the term “comprising” shall not be narrowlyconstrued as being limited to “consisting of” only, if not specificallymentioned. Rather, in the context of the present invention, “consistingof” is an embodiment specifically contemplated by the inventors to fallunder the scope of “comprising”, wherever “comprising” is used herein.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

BRIEF DESCRIPTION OF THE FIGURES

The figures shown in the following are merely illustrative and shalldescribe the present invention in a further way. These figures shall notbe construed to limit the present invention thereto.

FIG. 1 : G/C optimized mRNA sequence of R1691 coding for RSV-F proteinof the RSV long strain (RSV-F long) as comprised in the RSV-F long mRNAvaccine (SEQ ID NO. 31).

FIG. 2 : G/C optimized mRNA sequence of R2510 coding for RSV-F proteinof the RSV long strain (RSV-F long) as comprised in the RSV-F long mRNAvaccine (SEQ ID NO. 32).

FIG. 3 : G/C optimized mRNA sequence of R2821 coding for RSV-Fdel554-574 mutant protein of the RSV long strain (RSV-F long) (SEQ IDNO. 33).

FIG. 4 : G/C optimized mRNA sequence of R2831 coding for RSV-N proteinof the RSV long strain (RSV-F long) (SEQ ID NO. 34).

FIG. 5 : G/C optimized mRNA sequence of R2833 coding for RSV-M₂₋₁protein of the RSV long strain (RSV-F long) (SEQ ID NO. 35).

FIGS. 6A-C: show that the RSV-F long mRNA vaccine induces antibodytiters against the RSV-F protein comparable to those against inactivatedRSV.

-   -   Female BALB/c mice were intradermally (i.d.) injected with the        RSV-F long mRNA vaccine (160 μg of R1691) or Ringer-Lactate        (RiLa buffer) as buffer control. One group was intramuscularly        (i.m.) injected with 10 μg of the inactivated RSV long vaccine.        All animals received boost injections on days 21 and 35, blood        samples were collected on day 49 for the determination of        antibody titers of pooled sera as described in Example 2.    -   As can be seen, the RSV-F long mRNA vaccine induces anti-F        protein antibodies of the IgG1 and IgG2a subclasses. Antibody        titers are displayed in the graph (n=5 mice/group).

FIG. 7 : shows that the RSV-F long mRNA vaccine (R1691) induces along-lived immune response in mice.

-   -   The experiment was performed as described in Example 3 and        antibody total IgG titers determined by ELISA.    -   As can be seen, the antibody titers are stable for at least        eleven months after the last boost vaccination.

FIGS. 8A-D: show that the RSV-F long mRNA vaccine (R1691) induces RSVFusion (F) protein-specific multifunctional CD8⁺ T cells in mice.

-   -   The experiment was performed as described in Example 4 and T        cells were analysed by intracellular cytokine staining for the        antigen-specific induction of cytokines. Cells were stimulated        with a RSV-F peptide (stim. F; KYKNAVTEL) or a control influenza        HA peptide (stim. HA; IYSTVASSL). The line in the graph        represents the median value (n=5 mice/group).    -   As can be seen, the RSV-F long mRNA vaccine induces RSV        Fusion (F) protein-specific multifunctional CD8⁺ T cells in        contrast to the vaccine based on inactivated RSV which is not        able to induce F protein-specific CD8⁺ T cells.

FIGS. 9A-C: show that the RSV-N mRNA vaccine (R2831) inducesNucleoprotein (N)-specific multifunctional CD8⁺ T cells in mice.

-   -   The experiment was performed as described in Example 5 and T        cells were analysed by intracellular cytokine staining for the        antigen-specific induction of cytokines after stimulation with        ProMix RSV-N(15mer peptides). The line in the graph represents        the median value (n=5 mice/group).    -   As can be seen, the RSV-N mRNA vaccine induces RSV Nucleoprotein        (N)-specific multifunctional CD8⁺ T cells in contrast to the        vaccine based on inactivated RSV which is not able to induce N        protein-specific CD8⁺ T cells.

FIGS. 10A-C: show that the RSV-N mRNA vaccine (R2831) inducesNucleoprotein (N)-specific multifunctional CD4⁺ T cells in mice.

-   -   The experiment was performed as described in Example 5 and T        cells were analysed by intracellular cytokine staining for the        antigen-specific induction of cytokines after stimulation with        ProMix RSV-N(15mer peptides). The line in the graph represents        the median value (n=5 mice/group).    -   As can be seen, the RSV-N mRNA vaccine induces RSV Nucleoprotein        (N)-specific multifunctional CD4⁺ T cells in contrast to the        vaccine based on inactivated RSV which is not able to induce N        protein-specific CD4⁺ T cells.

FIGS. 11A-C: show that the RSV-M₂₋₁ mRNA vaccine (R2833) inducesM₂₋₁-specific multifunctional CD8⁺ T cells in mice.

-   -   The experiment was performed as described in Example 5 and T        cells were analysed by intracellular cytokine staining for the        antigen-specific induction of cytokines after stimulation with a        M₂₋₁ specific 9mer peptide. The line in the graph represents the        median value (n=5 mice/group).    -   As can be seen, the RSV-M₂₋₁ mRNA vaccine induces RSV        RSV-M₂₋₁-specific multifunctional CD8⁺ T cells in contrast to        the vaccine based on inactivated RSV which is not able to induce        M₂₋₁ protein-specific CD8⁺ T cells.

FIG. 12 : shows that RSV-F mRNA vaccines either alone (RSV-F=R2510;RSV-Fdel554-574 mutant=R2821) or in combination with mRNAs encodingother RSV proteins (RSV-N=R2831; RSV-M₂₋₁=R2833) induce humoral immuneresponses in cotton rats.

-   -   The experiment was performed as described in Example 6 and RSV-F        specific total IgG antibody titers were determined by ELISA on        day 49. Serum was analyzed in different dilution, as given below        the graph.

FIG. 13 : shows that RSV-F mRNA vaccines either alone (RSV-F, R2510;RSV-Fdel554-574 mutant, R2821) or in combination with mRNAs encodingother RSV proteins (RSV-N=R2831; RSV-M₂₋₁=R2833) induce the formation offunctional antibodies in cotton rats as shown by virus neutralizingantibody titers.

-   -   The experiment was performed as described in Example 6 and virus        neutralizing titers on day 49 were determined by plaque        reduction assay.

FIGS. 14A-B: show that RSV-F mRNA vaccines either alone (RSV-F=R2510;RSV-Fdel554-574 mutant=R2821) or in combination with mRNAs encodingother RSV proteins (RSV-N=R2831; RSV-M₂₋₁=R2833) reduce lung and nasaltiters in cotton rats challenged with RSV virus.

-   -   The experiment was performed as described in Example 6.    -   (A) Lung titers on day 5 after RSV challenge infection. All        animal groups vaccinated with mRNA vaccines showed virus titers        below the level of detection of the performed virus titration        demonstrating protection of vaccinated cotton rats in terms of        viral lung titers. In comparison to the mRNA vaccines the        vaccine based on formalin-inactivated virus were not able to        prevent virus titers in the lung.    -   (B) Nasal titers on day 5 after RSV challenge infection. The        viral titer in the nasal tissue was also strongly reduced in        groups vaccinated with mRNA. In comparison to the mRNA vaccines        the vaccine based on formalin-inactivated virus were not able to        reduce the nasal virus titers.

FIG. 15 : shows the results of the lung histopathology analysis from theRSV cotton rat challenge study described in Example 6.

FIGS. 16A-D: show the results of the quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) of viral genome copy numbers (bymeasuring copy numbers of the RSV NS-1 gene) or expressed cytokines fromlung tissue of the RSV infected animals (or controls) of the RSV cottonrat challenge study described in Example 6.

FIGS. 17A-B: show that RSV-F mRNA vaccines (RSV-F=R2510; RSV F*(RSV-Fdel554-574 mutant)=R2821) reduce lung titers in cotton ratschallenged with RSV virus.

-   -   The experiment was performed as described in Example 7.    -   (A) Lung titers on day 5 after RSV challenge infection. All        animal groups vaccinated intradermally with mRNA vaccines showed        reduced virus titers compared to the buffer control group        demonstrating protection of vaccinated cotton rats in terms of        viral lung titers. Already one single dose of RSV-F mRNA        vaccines efficiently reduced viral titers in the lung.    -   (B) Lung titers on day 5 after RSV challenge infection. The        viral titer in the lung was also strongly reduced in groups        intramuscularly vaccinated with mRNA.

EXAMPLES

The examples shown in the following are merely illustrative and shalldescribe the present invention in a further way. These examples shallnot be construed to limit the present invention thereto.

Example 1: Preparation of the mRNA Vaccine

1. Preparation of DNA and mRNA Constructs

For the present examples DNA sequences, encoding the RSV-F protein(R1691 and R2510), the RSV-F del554-574 (R2821) mutant protein, the RSVN-protein (R2831) and the RSV M2-1 protein (R2833) of the RSV longstrain were prepared and used for subsequent in vitro transcriptionreactions. The RSV-Fdel554-574 mutant protein has been describedpreviously (Oomens et al. 2006. J. Virol. 80(21):10465-77).

According to a first preparation, the DNA sequences coding for the abovementioned mRNAs were prepared. The construct R1691 was prepared bymodifying the wild type coding sequence by introducing a GC-optimizedsequence for stabilization, followed by a stabilizing sequence derivedfrom the alpha-globin-3′-UTR (muag (mutated alpha-globin-3′-UTR)according to SEQ ID No. 29), a stretch of 64 adenosines(poly(A)-sequence), a stretch of 30 cytosines (poly(C)-sequence), and ahistone stem loop according to SEQ ID No. 30. In SEQ ID NO: 31 (see FIG.1 ) the sequence of the corresponding mRNA is shown. The constructsR2510, R2821, R2831 and R2833 were prepared by introducing a 5′-TOP-UTRderived from the ribosomal protein 32L according to SEQ ID No. 23,modifying the wild type coding sequence by introducing a GC-optimizedsequence for stabilization, followed by a stabilizing sequence derivedfrom the albumin-3′-UTR (albumin7 according to SEQ ID No. 25), a stretchof 64 adenosines (poly(A)-sequence), a stretch of 30 cytosines(poly(C)-sequence), and a histone stem loop according to SEQ ID No. 30.In SEQ ID NOs: 32-35 (see FIG. 2-5 ) the sequences of the correspondingmRNAs are shown.

TABLE 1 mRNA constructs RNA Antigen Figure SEQ ID NO. R1691 RSV F 1 SEQID NO. 31 R2510 RSV F 2 SEQ ID NO. 32 R2821 RSV Fdel554-574 3 SEQ ID NO.33 R2831 RSVN 4 SEQ ID NO. 34 R2833 RSV M₂₋₁ 5 SEQ ID NO. 35

2. In Vitro Transcription

The respective DNA plasmids prepared according to paragraph 1 weretranscribed in vitro using T7 polymerase in the presence of a CAP analog(m⁷GpppG). Subsequently the mRNA was purified using PureMessenger®(CureVac, Tubingen, Germany; WO2008/077592A1).

3. Reagents

Complexation Reagent: protamine

4. Preparation of the Vaccine

The mRNA was complexed with protamine by addition of protamine to themRNA in the ratio (1:2) (w/w) (adjuvant component). After incubation for10 minutes, the same amount of free mRNA used as antigen-providing RNAwas added.

For example: RSV-F long vaccine (R1691): comprising an adjuvantcomponent consisting of mRNA coding for RSV F protein long (R1691)according to SEQ ID NO. 31 complexed with protamine in a ratio of 2:1(w/w) and the antigen-providing free mRNA coding for RSV F protein long(R1691) according to SEQ ID NO. 31 (ratio 1:1; complexed RNA:free RNA).

Example 2: Induction of a Humoral Immune Response by the RSV-F Long mRNAVaccine in Mice

Immunization

On day zero, BALB/c mice were intradermally (i.d.) injected with theRSV-F long mRNA vaccine (R1691 according to Example 1; 25μg/mouse/vaccination day) or Ringer-lactate (RiLa) as buffer control asshown in Table 2. A control group was intramuscularly (i.m.) injectedwith 10 μg of the inactivated RSV long vaccine. The inactivated“Respiratory Syncytial Virus Antigen” (inactivated RSV long) waspurchased from the INSTITUT VIRION/SERION GmbH-SERION IMMUNDIAGNOSTICAGmbH. The inactivated virus was diluted in sterile PBS, so that a finalconcentration of 0.2 μg/μL was achieved. All animals received boostinjections on day 14 and day 28. Blood samples were collected on day 42for the determination of anti-RSV F antibody titers.

TABLE 2 Animal groups Strain No. Route Vaccine Group sex mice volumedose Vaccination schedule 1 BALB/c 5 i.d. R1691 d0: prime, d14: boost,Female 2 × 50 μl 2 μg d28: boost, d42: blood collection 2 BALB/c 5 i.m.Inactivated d0: prime, d14: boost, Female 2 × 25 μl RSV long d28: boost,10 μg d42: blood collection 3 BALB/c 5 i.d. 80% Ringer d0: prime, d14:boost, Female 2 × 50 μl Lactate (RiLa) d28: boost, buffer d42: bloodcollection

Determination of Anti-RSV F Protein Antibodies by ELISA

ELISA plates are coated with recombinant human RSV fusion glycoprotein(rec.hu F-protein, final conc.: 5 μg/mL) (Sino Biological Inc.). Coatedplates are incubated using given serum dilutions and binding of specificantibodies to the F protein is detected using biotinylated isotypespecific anti-mouse antibodies in combination with streptavidin-HRP(horse radish peroxidase) with ABTS substrate.

Results

As can be seen in FIG. 6 , the RSV-F long mRNA vaccine induces antibodytiters (total IgG, IgG1 and IgG2a) against the RSV F protein comparableto those against inactivated RSV.

Example 3: Induction of a Long-Lived Humoral Immune Response by theRSV-F Long mRNA Vaccine in Mice

Immunization

BALB/c mice were intradermally (i.d.) injected with 20 μg of the RSV-Flong mRNA vaccine (R1691) or Ringer Lactate (RiLa) buffer according tothe vaccination schedule shown in Table 3. Blood was collected 2 weeks,4 months and 11 months after the last immunization.

TABLE 3 Animal groups Strain Number Route Vaccine Vaccination scheduleGroup sex of mice volume dose (day) 1 BALB/c 5 i.d. R1691 d0, d14, d28Female 100 μl 20 μg 4 BALB/c 5 i.d. 80% RiLa d0, d14, d28 Female 100 μlbuffer

Results

As can be seen in FIG. 7 , the RSV-F long mRNA vaccine induced along-lived immune response as demonstrated by stable antibody titers forat least 11 months after the last boost vaccination.

Example 4: Induction of a Cellular Immune Response by the RSV-F LongmRNA Vaccine in Mice

Immunization

On day zero, BALB/c mice were intradermally (i.d.) injected with theRSV-F long mRNA vaccine R1691 (20 μg/mouse/vaccination day) orRinger-lactate (RiLa) as buffer control as shown in Table 4. A controlgroup was intramuscularly (i.m.) injected with 10 μg of the inactivatedRSV long vaccine. The inactivated “Respiratory Syncytial Virus Antigen”(inactivated RSV long) was purchased from the INSTITUT VIRION/SERIONGmbH-SERION IMMUNDIAGNOSTICA GmbH. The inactivated virus was diluted insterile PBS, so that a final concentration of 0.2 μg/μL was achieved.

All animals received boost injections on days 14 and 28. Spleens werecollected on day 34 for the analysis of antigen-specific T cells.

TABLE 4 Animal groups Strain Number Route Vaccine Vaccination Group sexof mice volume dose schedule 1 BALB/c 5 i.d. R1691 d0: prime, d14:boost, Female 2 × 50 μl 20 μg d28: boost, d34: spleen collection 2BALB/c 5 i.m. Inactivated d0: prime, d14: boost, Female 2 × 25 μl RSVlong d28: boost, 10 μg d34: spleen collection 3 BALB/c 5 i.d. Ringer d0:prime, d14: boost, Female 2 × 50 μl Lactate d28: boost, (RiLa) d34:spleen collection buffer

Intracellular Cytokine Staining

Splenocytes from vaccinated and control mice were isolated according toa standard protocol. Briefly, isolated spleens were grinded through acell strainer and washed in PBS/1% FBS followed by red blood cell lysis.After an extensive washing step with PBS/1% FBS splenocytes were seededinto 96-well plates (2×10⁶ cells/well). The next day cells werestimulated with a RSV-F peptide (KYKNAVTEL; 5 μg/ml; H-2kd-restructedT-cell epitope) or an irrelevant control peptide derived from theinfluenza HA protein (IYSTVASSL; 5 μg/ml; purchased from EMCMicrocollections) and 2.5 μg/ml of an anti-CD28 antibody (BDBiosciences) for 6 hours at 37° C. in the presence of the mixture ofGolgiPlug™/GolgiStop™ (Protein transport inhibitors containing BrefeldinA and Monensin, respectively; BD Biosciences). After stimulation cellswere washed and stained for intracellular cytokines using theCytofix/Cytoperm reagent (BD Biosciences) according to themanufacturer's instructions. The following antibodies were used forstaining: CD8-PECy7 (1:200), CD3-FITC (1:200), IL2-PerCP-Cy5.5 (1:100),TNFα-PE (1:100), IFNγ-APC (1:100) (eBioscience), CD4-BD Horizon V450(1:200) (BD Biosciences) and incubated with Fcγ-block diluted 1:100.Aqua Dye was used to distinguish live/dead cells (Invitrogen). Cellswere collected using a Canto II flow cytometer (Beckton Dickinson). Flowcytometry data were analysed using FlowJo software (Tree Star, Inc.).Statistical analysis was performed using GraphPad Prism software,Version 5.01. Statistical differences between groups were assessed bythe Mann Whitney test.

Results

As can be seen from FIG. 8 , the RSV-F long mRNA vaccine (R1691) inducedIFNγ positive, TNFα positive and IFNγ/TNFα double-positivemultifunctional CD8⁺ T cells directed against RSV F protein.Surprisingly the vaccine based on inactivated RSV virus was not able toinduce antigen-specific CD8⁺ T cells.

Example 5: Induction of Cellular Immune Responses by the RSV-N andRSV-M₂₋₁ mRNA Vaccines in Mice

Immunization

On day zero, BALB/c mice were intradermally (i.d.) injected withdifferent doses of the RSV-N mRNA vaccine R2831, the RSV-M₂₋₁ mRNAvaccine R2833 or Ringer-lactate (RiLa) as buffer control as shown inTable 5. A control group was intramuscularly (i.m.) injected with 10 μgof the inactivated RSV long vaccine. The inactivated “RespiratorySyncytial Virus Antigen” (inactivated RSV long) was purchased from theINSTITUT VIRION/SERION GmbH-SERION IMMUNDIAGNOSTICA GmbH. Theinactivated virus was diluted in sterile PBS, so that a finalconcentration of 0.2 μg/μL was achieved. All animals received boostinjections on days 7 and 21. Spleens were collected on day 27 for theanalysis of antigen-specific T cells.

TABLE 5 Animal groups Strain No. Route Vaccine Group sex mice volumedose Vaccination schedule 1 BALB/c 5 i.d. R2831 RSV-N d0: prime, d7:boost, d21: boost, Female 1 × 50 μl 40 μg d27: spleen collection 2BALB/c 5 i.d. R2831 RSV-N d0: prime, d7: boost, d21: boost, Female 1 ×25 μl 20 μg d27: spleen collection 3 BALB/c 5 i.d. R2831 RSV-N d0:prime, d7: boost, d21: boost, Female 1 × 12.5 10 μg d27: spleencollection μl 4 BALB/c 5 i.d. R2833 RSV-M₂₋₁ d0: prime, d7: boost, d21:boost, Female 1 × 50 μl d27: spleen collection 40 μg 5 BALB/c 5 i.d.R2833 RSV-M₂₋₁ d0: prime, d7: boost, d21: boost, Female 1 × 25 μl d27:spleen collection 20 μg 6 BALB/c 5 i.d. R2833 RSV-M₂₋₁ d0: prime, d7:boost, d21: boost, Female 1 × 12.5 d27: spleen collection μl 10 μg 7BALB/c 5 i.m. Inactiv. RSV d0: prime, d7: boost, d21: boost, Female 2 ×25 μl long d27: spleen collection 10 μg 8 BALB/c 5 i.d. 100% RiLa d0:prime, d7: boost, d21: boost, Female 1 × 50 μl buffer d27: spleencollection

Intracellular cytokine staining was performed as described in Example 4except that cells were treated with the following stimulators at:

M2-1 peptide (5 μg/ml) group 4 to 8; (SYIGSINNI from Prolmmune); ProMixN (1 μg/ml) 1-3, group 7 and 8; (PX39 from Proimmune); control:medium+DMSO+anti-CD28, group 1-8 as descripted above.

Results

As can be seen from FIG. 9 , the RSV-N mRNA vaccine (R2831) induced IFNγpositive, TNFα positive and IFNγ/TNFα double-positive multifunctionalCD8⁺ T cells directed against RSV N protein in mice.

Surprisingly the vaccine based on inactivated RSV virus was not able toinduce antigen-specific CD8⁺ T cells.

As can be seen from FIG. 10 , the RSV-N mRNA vaccine (R2831) inducedIFNγ positive, TNFα positive and IFNγ/TNFα double-positivemultifunctional CD4⁺ T cells directed against RSV N protein in mice.

Surprisingly the vaccine based on inactivated RSV virus was not able toinduce antigen-specific CD4⁺ T cells.

As can be seen from FIG. 11 , the RSV-M₂₋₁ mRNA vaccine (R2833) inducedIFNγ positive, TNFα positive and IFNγ/TNFα double-positivemultifunctional CD8⁺ T cells directed against RSV M₂₋₁ protein in mice.

Surprisingly the vaccine based on inactivated RSV virus was not able toinduce antigen-specific CD8⁺ T cells.

Example 6: RSV Cotton Rat Challenge Study I

For the development of RSV vaccines the cotton rat is an accepted animalmodel, especially for the challenge infection. Cotton rats respond toformalin-inactivated RSV virus vaccine preparations with enhanced lungpathology. This allows the evaluation of the safety of a vaccination interms of enhanced disease phenomenon.

To broaden and optimize the RSV-specific immune response, mRNA vaccinesencoding different RSV proteins (RSV F, mutant RSV-Fdel554-574, N andM₂₋₁) were prepared according to Example 1. In order to assess theeffect of single or combined vaccines, these vaccines were administeredeither alone or in combination (cocktail vaccine) as shown in Table 5.Vaccine volumes of 2×50 μl were injected intradermally (i.d.) into theback skin of cotton rats. Additional groups were immunizedintramuscularly (i.m.) with β-propiolactone inactivated RSV (INSTITUTVIRION/SERION GmbH-SERION IMMUNDIAGNOSTICA GmbH), formalin-inactivatedRSV (Sigmovir) or live RSV/A2 (Sigmovir) (10⁵ plaque forming units, pfu)to compare their immunogenicity to mRNA vaccines. Another group receivedi.m. injections of the monoclonal anti-RSV antibody SYNAGIS®(Palivizumab) as passive immunization. SYNAGIS® was administered with adose of 15 mg/kg on the day prior to RSV challenge infection. Thereforethe animals were weighed and the appropriate amount of SYNAGIS® wascalculated according to the animals' weight. The maximal volume for i.m.injection was 200 μl per 100 g rat. After immunization the cotton ratswere challenged by intranasal (i.n.) infection with RSV/A2 virus (10⁵PFU in 100 μl; Sigmovir).

TABLE 5 Animal groups Vaccine # of N per Vaccination Bleed Groups doseVolume Antigen Route administrations group (day) (day) A β- 100 μl — IM3 5 0, 14, 28 14, 28, 49 propiolactone inactivated RSV 20 μg B R2510 2 ×50 μl RSV F ID 3 5 0, 14, 28 14, 28, 49 80 μg C R2821 2 × 50 μl RSV F ID3 5 0, 14, 28 14, 28, 49 80 μg mutant D R2510 + 2 × 50 μl 3 5 0, 14, 2814, 28, 49 R2831 RSV F + ID “cocktail I” RSVN each 40 μg E R2510 + 2 ×50 μl RSV F + ID 3 5 0, 14, 28 14, 28, 49 R2833 RSV “cocktail II” M₂₋₁each 40 μg F R2510 + 2 × 50 μl RSV F + ID 3 5 0, 14, 28 14, 28, 49R2831 + R2833 RSV “cocktail III” M₂₋₁ + each 26.666 μg RSV N G RiLa 2 ×50 μl — ID 3 5 0, 14, 28 14, 28, 49 H FI-RSV 100 μl — IM 2 5 0, 28 28,49 Lot#100 (diluted 1:100 in PBS) I Live RSV/A2 100 μl — IM 1 5  0 4910⁵ pfu J SYNAGIS ® — IM 1 5 48 49 (15 mg/kg) K Neg. control — N/A N/A 5— —

The following assays were performed to analyze the immune responses: RSVF-protein serum IgG ELISA, RSV virus neutralizing antibody titers (VNT),RSV viral titrations and pulmonary histopathology.

RSV F-Protein Serum IgG ELISA

The induction of anti-RSV F protein antibodies were determined by ELISAaccording to Example 2.

RSV Virus Neutralizing Antibody Titers (VNT)

Sera were analysed by the virus neutralization test (VNT). Briefly, serasamples were diluted 1:10 with EMEM, heat inactivated and seriallydiluted further 1:4. Diluted sera samples were incubated with RSV (25-50PFU) for 1 hour at room temperature and inoculated in duplicates ontoconfluent HEp-2 monolayers in 24 well plates. After one hour incubationat 37° C. in a 5% CO₂ incubator, the wells were overlayed with 0.75%Methylcellulose medium. After 4 days of incubation, the overlay wasremoved and the cells were fixed with 0.1% crystal violet stain for onehour and then rinsed and air dried. The corresponding reciprocalneutralizing antibody titers were determined at the 60% reductionend-point of the virus control.

RSV Viral Titrations and Pulmonary Histopathology

On day 54 nasal tissue was harvested and homogenized for viraltitrations. The lung was harvested en bloc and tri-sected for viraltitration (left section), histopathology (right section), and PCRanalysis (lingular lobe). In addition, RSV viral genome copy numbers (bymeasuring copy numbers of the RSV NS-1 gene) and cytokine mRNA levelswere determined by quantitative reverse transcription polymerase chainreaction (qRT-PCR).

Results

As can be seen from FIG. 12 , the RSV-F mRNA vaccines either alone(RSV-F=R2510; RSV-Fdel554-574 mutant=R2821) or in combination with mRNAsencoding other RSV proteins (RSV-N=R2831; RSV-M2-1=R2833), induce RSV Fspecific humoral immune responses in cotton rats as shown by total IgGantibody titers on day 49.

As can be seen from FIG. 13 , the RSV-F mRNA vaccines either alone(RSV-F=R2510; RSV-Fdel554-574 mutant=R2821) or in combination with mRNAsencoding other RSV proteins (RSV-N, R2831=RSV-M2-1=R2833), induce theformation of RSV specific functional antibodies in cotton rats as shownby virus neutralizing antibody titers.

As can be seen from FIG. 14 , the RSV-F mRNA vaccines either alone(RSV-F=R2510; RSV-Fdel554-574 mutant=R2821) or in combination with mRNAsencoding other RSV proteins (RSV-N=R2831; RSV-M2-1=R2833), reduce lungand nasal viral titers in cotton rats challenged with RSV virus.

As can be seen in FIG. 14A, all animal groups vaccinated with mRNAvaccines showed virus titers below the level of detection of theperformed virus titration demonstrating protection of vaccinated cottonrats in terms of viral lung titers. By contrast, theFormalin-inactivated virus vaccine reduced only minimally the lung virustiter compared to the RiLa buffer control group. The effect of theβ-propiolactone inactivated RSV vaccine was more pronounced but did notreduce the virus lung titer below the detection level in all animals ofthis group. As can be seen in FIG. 14B, the viral titer in the nasaltissue was also strongly reduced in groups vaccinated with mRNA. Nasalviral titers of the Formalin-inactivated virus were comparable to theviral titer in the RiLa vaccinated group. The β-propiolactoneinactivated virus vaccine was more effective (at least for two of fiveanimals). In contrast thereto, all mRNA vaccinated groups had reducednasal virus titer compared to RiLa vaccinated group.

As can be seen from FIG. 15 , the lung histopathology analysis from theRSV cotton rat challenge study reveals different pathology scores forthe various animal groups. From the histopathology it can be concludedthat none of the mRNA vaccinated groups displayed enhanced lungpathology as it is the case for the group that was vaccinated using theFormalin-inactivated RSV vaccine. The average pathology scores forperibronchiolitis (PB), perivasculitis (PV), insterstitial pneumonia(IP) and alveolitis (A) are much lower for all groups vaccinated withmRNA compared to group H (Formalin-inactivated RSV). In addition thegroups being vaccinated with R2510 (group B; RSV F) or R2821 (group C;RSV F mutant) seem to exhibit reduced lung pathology compared to theRiLa buffer vaccinated and subsequently RSV infected group (G).

As can be seen in FIG. 16 , the quantitative RT-PCR reveals differentexpression patterns for the various animal groups. The quantification ofRSV genome copies by measuring the RSV NS-1 gene is displayed in A.Genome copy numbers are reduced by vaccination using mRNA vaccinescompared to the RiLa buffer control (group G). This is not the case forthe group that was vaccinated using formalin-inactivated-RSV (group H).As it is shown in B, the vaccination using the formalin-inactivated-RSVvaccine (group H) induces enhanced expression of the TH2 cytokine IL-4compared to the control group that was vaccinated with RiLa buffer(group G). By contrast, the vaccination with mRNA R2821 encoding theRSV-F mutant significantly reduced IL-4 mRNA expression compared to theRiLa control group in the lung after RSV challenge infection. C.Expression of INF-γ□mRNA. D. Expression of IL-5 mRNA. The expression ofIL-5 is significant reduced in groups vaccinated using R2510 or R2821compared to RiLa buffer vaccinated animals. The expression of the viralNS-1 RNA or cytokine mRNAs, which were isolated from lung tissue, ismeasured on day 5 post-challenge. The statistical analysis was performedwith the student T-test (*p<0.05 when compared to group G (RiLacontrol)).

Example 7: RSV Cotton Rat Challenge Study II

mRNA vaccines encoding RSV F protein (F) or mutant RSV-F protein (F*)(RSV F del554-574) were prepared according to Example 1. In order toassess the effect of single or several vaccinations (prime and boostvaccinations), these vaccines were administered once, twice or 3 times(as shown in Table 6). Vaccine volumes of 2×50 μl were injectedintradermally (i.d.) into the back skin of cotton rats. Additionalgroups were immunized intramuscularly (i.m.) with vaccine volumes of1×100 μl into the right hind leg. As a control, one group was injectedintradermally with Ringer-Lactate buffer (buffer). After immunization,the cotton rats were challenged by intranasal (i.n.) infection withRSV/A2 virus (10⁵ PFU in 100 μl; Sigmovir). As a control, one group wasnot treated and remained unchallenged with virus (untreated).

TABLE 5 Animal groups Vaccine # of N per Vaccination Challenge Groupsdose Volume Antigen Route administrations group (day) (day) F* i.d. 3×R2821 2 × 50 μl RSV F ID 3 5 0, 14, 28 49 80 μg mutant F* i.d. 2× R28212 × 50 μl RSV F ID 2 5 0,14 49 80 μg mutant F* i.d. 1× R2821 2 × 50 μlRSV F ID 1 5 0 49 80 μg mutant Fi.d. 3× R2510 2 × 50 μl RSV F ID 3 5 0,14, 28 49 80 μg Fi.d. 2× R2510 2 × 50 μl RSV F ID 2 5 0, 14 49 80 μgFi.d. 1× R2510 2 × 50 μl RSV F ID 1 5 0 49 80 μg F* i.m. R2821 1 × 100μl RSV F IM 2 5 0, 14 49 80 μg mutant F i.m. R2510 1 × 100 μl RSV F IM 25 0, 14 49 80 μg Buffer — 2 × 50 μl ID 3 5 0, 14, 28 49 untreated — —N/A N/A 5 — —

RSV Viral Titrations

The determination of RSV viral titers was conducted as described inExample 6.

Results

As shown in FIG. 17A, already one single intradermal vaccination withmRNA vaccines coding for RSV F protein (F) or mutant RSV-F protein (F*)(RSV F del554-574) was highly efficient in reducing the viral titer inthe lung compared to the buffer control group. A second and thirdvaccination (“boost vaccinations) reduced the viral titers belowdetection level.

As shown in FIG. 17B, already two intramuscular vaccinations with mRNAvaccines coding for RSV F protein (F) or mutant RSV-F protein (F*) (RSVF del554-574) strongly reduced the viral titer in the lung compared tothe buffer control group.

1. mRNA sequence comprising a coding region, encoding at least oneantigenic peptide or protein derived from the fusion protein F, theglycoprotein G, the short hydrophobic protein SH, the matrix protein M,the nucleoprotein N, the large polymerase L, the M2-1 protein, the M2-2protein, the phosphoprotein P, the non-structural protein NS1 or thenon-structural protein NS2 of Respiratory syncytial virus (RSV), or afragment, variant or derivative thereof; wherein the G/C content of thecoding region is increased compared with the G/C content of the codingregion of the wild type mRNA, and wherein the coded amino acid sequenceof said GC-enriched mRNA is preferably not being modified compared withthe coded amino acid sequence of the wild type mRNA.
 2. The mRNAsequence according to claim 1, wherein the coding region encodes thefull-length protein of fusion protein F, nucleoprotein N or glycoproteinG of Respiratory syncytial virus (RSV).
 3. The mRNA sequence accordingto any of claims 1 to 2, wherein the antigenic peptide or protein isderived from the RSV strain ATCC VR-26 long.
 4. The mRNA sequenceaccording to any of claims 1 to 3 comprising additionally a) a 5′-CAPstructure, b) a poly(A) sequence, c) and optionally a poly (C) sequence.5. The mRNA sequence according to claim 4, wherein the poly(A) sequencecomprises a sequence of about 25 to about 400 adenosine nucleotides,preferably a sequence of about 50 to about 400 adenosine nucleotides,more preferably a sequence of about 50 to about 300 adenosinenucleotides, even more preferably a sequence of about 50 to about 250adenosine nucleotides, most preferably a sequence of about 60 to about250 adenosine nucleotides.
 6. The mRNA sequence according to any ofclaims 1 to 5 comprising additionally at least one histone stem-loop. 7.The mRNA sequence according to claim 6, wherein the at least one histonestem-loop is selected from following formulae (I) or (II): formula (I)(stem-loop sequence without stem bordering elements):

formula (II) (stem-loop sequence with stem bordering elements):

wherein: stem1 or stem2 bordering elements is a consecutive sequence of1 to 6, preferably N₁₋₆ of 2 to 6, more preferably of 2 to 5, even morepreferably of 3 to 5, most preferably of 4 to 5 or 5 N, wherein each Nis independently from another selected from a nucleotide selected fromA, U, T, G and C, or a nucleotide analogue thereof; stem1 [N₀₋₂GN₃₋₅] isreverse complementary or partially reverse complementary with elementstem2, and is a consecutive sequence between of 5 to 7 nucleotides;wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of 0 to 1,more preferably of 1 N, wherein each N is independently from anotherselected from a nucleotide selected from A, U, T, G and C or anucleotide analogue thereof; wherein N₃₋₅ is a consecutive sequence of 3to 5, preferably of 4 to 5, more preferably of 4 N, wherein each N isindependently from another selected from a nucleotide selected from A,U, T, G and C or a nucleotide analogue thereof, and wherein G isguanosine or an analogue thereof, and may be optionally replaced by acytidine or an analogue thereof, provided that its complementarynucleotide cytidine in stem2 is replaced by guanosine; loop sequence[N₀₋₄(U/T)N₀₋₄] is located between elements stem1 and stem2, and is aconsecutive sequence of 3 to 5 nucleotides, more preferably of 4nucleotides; wherein each N₀₋₄ is independent from another a consecutivesequence of 0 to 4, preferably of 1 to 3, more preferably of 1 to 2 N,wherein each N is independently from another selected from a nucleotideselected from A, U, T, G and C or a nucleotide analogue thereof; andwherein U/T represents uridine, or optionally thymidine; stem2[N₃₋₅CN₀₋₂] is reverse complementary or partially reverse complementarywith element stem1, and is a consecutive sequence between of 5 to 7nucleotides; wherein N₃₋₅ is a consecutive sequence of 3 to 5,preferably of 4 to 5, more preferably of 4 N, wherein each N isindependently from another selected from a nucleotide selected from A,U, T, G and C or a nucleotide analogue thereof; wherein N₀₋₂ is aconsecutive sequence of 0 to 2, preferably of 0 to 1, more preferably of1 N, wherein each N is independently from another selected from anucleotide selected from A, U, T, G and C or a nucleotide analoguethereof; and wherein C is cytidine or an analogue thereof, and may beoptionally replaced by a guanosine or an analogue thereof provided thatits complementary nucleotide guanosine in stem1 is replaced by cytidine;

wherein stem1 and stem2 are capable of base pairing with each otherforming a reverse complementary sequence, wherein base pairing may occurbetween stem1 and stem2, or forming a partially reverse complementarysequence, wherein an incomplete base pairing may occur between stem1 andstem2.
 8. The mRNA sequence according to claim 7, wherein the at leastone histone stem-loop is selected from at least one of followingformulae (Ia) or (IIa):

formula (Ia) (stem-loop sequence without stem bordering elements)

formula (IIa) (stem-loop sequence with stem bordering elements)
 9. ThemRNA sequence according to any of claims 1 to 8 comprising additionallya 3′-UTR element.
 10. The mRNA sequence according to claim 9, whereinthe at least one 3′UTR element comprises or consists of a nucleic acidsequence which is derived from a 3′UTR of a gene providing a stable mRNAor from a homolog, a fragment or a variant thereof.
 11. The mRNAsequence according to claim 10, wherein the 3′UTR element comprises orconsists of a nucleic acid sequence derived from a 3′UTR of a geneselected from the group consisting of an albumin gene, an α-globin gene,a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and acollagen alpha gene, or from a homolog, a fragment or a variant thereof.12. The mRNA sequence according to claim 11, wherein the 3′-UTR elementcomprises or consists of a nucleic acid sequence derived from a 3′UTR ofα-globin gene, preferably comprising the corresponding RNA sequence ofthe nucleic acid sequence according to SEQ ID NO. 29, a homolog, afragment, or a variant thereof;
 13. The mRNA sequence according to anyof claims 9 to 12; wherein the mRNA sequence comprises, preferably in5′- to 3′-direction: a.) a 5′-CAP structure, preferably m7GpppN; b.) acoding region encoding at least one antigenic peptide or protein ofRespiratory syncytial virus (RSV), preferably derived from the fusionprotein F of Respiratory syncytial virus (RSV); c.) a 3′-UTR elementcomprising or consisting of a nucleic acid sequence which is derivedfrom a alpha globin gene, preferably comprising the corresponding RNAsequence of the nucleic acid sequence according to SEQ ID NO. 29, ahomolog, a fragment or a variant thereof; d.) a poly(A) sequence,preferably comprising 64 adenosines; e.) a poly(C) sequence, preferablycomprising 30 cytosines; and f.) a histone-stem-loop, preferablycomprising the corresponding RNA sequence to the nucleic acid sequenceaccording to SEQ ID No
 30. 14. The mRNA sequence according to claim 13,wherein the mRNA sequence comprises the RNA sequence according to SEQ IDNo.
 31. 15. The mRNA sequence according to claim 9, wherein the at leastone 3′UTR element comprises or consist of a nucleic acid sequence whichis derived from the 3′UTR of a vertebrate albumin gene or from a variantthereof, preferably from the 3′UTR of a mammalian albumin gene or from avariant thereof, more preferably from the 3′UTR of a human albumin geneor from a variant thereof, even more preferably from the 3′UTR of thehuman albumin gene according to GenBank Accession number NM_000477.5, orfrom a fragment or variant thereof.
 16. The mRNA sequence according toclaim 15, wherein the 3′UTR element is derived from a nucleic acidsequence according to SEQ ID NO. 25, preferably from a corresponding RNAsequence, a homolog, a fragment or a variant thereof.
 17. The mRNAsequence according to any of claims 1 to 16 comprising additionally a5′-UTR element which comprises or consists of a nucleic acid sequencewhich is derived from the 5′UTR of a TOP gene preferably from acorresponding RNA sequence, a homolog, a fragment, or a variant thereof,preferably lacking the 5′TOP motif.
 18. The mRNA sequence according toclaim 17, wherein the 5′UTR element comprises or consists of a nucleicacid sequence which is derived from a 5′UTR of a TOP gene encoding aribosomal protein, preferably from a corresponding RNA sequence or froma homolog, a fragment or a variant thereof, preferably lacking the 5′TOPmotif.
 19. The mRNA sequence according to claim 18, wherein the 5′UTRelement comprises or consists of a nucleic acid sequence which isderived from a 5′UTR of a TOP gene encoding a ribosomal Large protein(RPL) or from a homolog, a fragment or variant thereof, preferablylacking the 5′TOP motif and more preferably comprising or consisting ofa corresponding RNA sequence of the nucleic acid sequence according toSEQ ID NO.
 23. 20. The mRNA sequence according to claim 19; wherein themRNA sequence comprises, preferably in 5′- to 3′-direction: a.) a 5′-CAPstructure, preferably m7GpppN; b.) a 5′-UTR element which comprises orconsists of a nucleic acid sequence which is derived from the 5′-UTR ofa TOP gene, preferably comprising or consisting of the corresponding RNAsequence of the nucleic acid sequence according to SEQ ID NO. 23, ahomolog, a fragment or a variant thereof; c.) a coding region encodingat least one antigenic peptide or protein of Respiratory syncytial virus(RSV), preferably derived from the fusion protein F of Respiratorysyncytial virus (RSV); d.) a 3′UTR element comprising or consisting of anucleic acid sequence which is derived from a gene providing a stablemRNA, preferably comprising or consisting of the corresponding RNAsequence of a nucleic acid sequence according to SEQ ID NO. 18, ahomolog, a fragment or a variant thereof; e.) a poly(A) sequencepreferably comprising 64 adenosines; f.) a poly(C) sequence, preferablycomprising 30 cytosines; and g.) a histone-stem-loop, preferablycomprising the corresponding RNA sequence of the nucleic acid sequenceaccording to SEQ ID No
 30. 21. The mRNA sequence according to claim 20,wherein the mRNA sequence comprises the RNA sequence according to SEQ IDNo. 32 or
 33. 22. The mRNA sequence according to claims 1 to 21, whereinthe mRNA sequence is associated with or complexed with a cationic orpolycationic compound or a polymeric carrier, optionally in a weightratio selected from a range of about 6:1 (w/w) to about 0.25:1 (w/w),more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), even morepreferably of about 4:1 (w/w) to about 1:1 (w:w) or of about 3:1 (w/w)to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w) toabout 2:1 (w/w) of mRNA to cationic or polycationic compound and/or witha polymeric carrier; or optionally in a nitrogen/phosphate ratio of mRNAto cationic or polycationic compound and/or polymeric carrier in therange of about 0.1-10, preferably in a range of about 0.3-4 or 0.3-1,and most preferably in a range of about 0.5-1 or 0.7-1, and even mostpreferably in a range of about 0.3-0.9 or 0.5-0.9.
 23. The mRNA sequenceaccording to claim 22, wherein the mRNA sequence is associated orcomplexed with a cationic protein or peptide, preferably protamine. 24.A composition comprising a plurality or more than one of mRNA sequenceseach according to any of claims 1 to
 23. 25. Pharmaceutical compositioncomprising an mRNA sequence as defined according to any of claims 1 to23 or a composition as defined according to claim 24 and optionally apharmaceutically acceptable carrier.
 26. Pharmaceutical compositionaccording to claim 25, wherein the mRNA sequence is complexed at leastpartially with a cationic or polycationic compound and/or a polymericcarrier, preferably cationic proteins or peptides and most preferablyprotamine.
 27. Pharmaceutical composition according to claim 26, whereinthe ratio of complexed mRNA to free mRNA is selected from a range ofabout 5:1 (w/w) to about 1:10 (w/w), more preferably from a range ofabout 4:1 (w/w) to about 1:8 (w/w), even more preferably from a range ofabout 3:1 (w/w) to about 1:5 (w/w) or 1:3 (w/w), and most preferably theratio of complexed mRNA to free mRNA is selected from a ratio of 1:1(w/w).
 28. Kit or kit of parts comprising the components of the mRNAsequence as defined according to any of claims 1 to 23, the compositionas defined according to claim 24, the pharmaceutical composition asdefined according to any of claims 25 to 27 and optionally technicalinstructions with information on the administration and dosage of thecomponents.
 29. mRNA sequence as defined according to any of claims 1 to23, composition as defined according to claim 24, pharmaceuticalcomposition as defined according to any of claims 25 to 27, and kit orkit of parts as defined according to claim 28 for use as a medicament.30. mRNA sequence as defined according to any of claims 1 to 23,composition as defined according to claim 24, pharmaceutical compositionas defined according to any of claims 25 to 27, and kit or kit of partsas defined according to claim 28 for use in the treatment or prophylaxisof RSV infections.
 31. mRNA sequence, composition, pharmaceuticalcomposition and kit or kit of parts for use according to claim 30,wherein the treatment is combined with administration of RSV immuneglobuline, particularly Palivizumab.
 32. A method of treatment orprophylaxis of RSV infections comprising the steps: a) providing themRNA sequence as defined according to any of claims 1 to 23, thecomposition as defined according to claim 24, the pharmaceuticalcomposition as defined according to any of claims 25 to 27, or the kitor kit of parts as defined according to claim 28; b) applying oradministering the mRNA sequence, the composition, the pharmaceuticalcomposition or the kit or kit of parts to a tissue or an organism; c)optionally administering RSV immune globuline.